Low volume chemical and biochemical reaction system

ABSTRACT

A method and device for preparing nanoscale reactions. An automated system utilizes an array of reaction chambers. The ends of the chambers are temporarily sealed with deformable membranes and reactions effected by incubation of temperature cycling. Reaction mixtures may be assembled by using the reaction containers to meter reaction components. After the reaction is finished, the reaction containers may be dispensed onto a substrate and the reaction products analyzed. An automated transfer device may be used for automated transport of the reaction container array or other transportable elements.

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application is a continuation of U.S. patent applicationSer. No. 09/577,199 filed May 23, 2000, and claims priority from U.S.provisional application No. 60/146,732 filed Aug. 2, 1999.

TECHNICAL FIELD OF THE INVENTION

[0002] This invention relates to a method and apparatus for performingsmall scale reactions. In particular, the instant disclosure pertains tosmall scale cycling reactions and devices for assembly of sub-microliterreaction mixtures.

BACKGROUND OF THE INVENTION

[0003] The Human Genome Program is a scientific endeavor which is anational priority of the United States. The original goal of thefederally funded U.S. effort had been to complete the sequence atten-fold coverage by the year 2005. A draft, five-fold deep version ofthe human genome will now be produced by the year 2001. To accomplishthis goal, the effort has accelerated to improve sequencing throughputrates and reduce DNA sequencing costs.

[0004] In the late 1970s, Sanger et al. developed an enzymatic chaintermination method for DNA sequence analysis that produces a nested setof DNA fragments with a common starting point and random terminations atevery nucleotide throughout the sequence. Lloyd Smith, Lee Hood, andothers modified the Sanger method to use four fluorescent labels insequencing reactions enabling single lane separations. This resulted inthe creation of the first automated DNA sequencers. More recently,fluorescent energy-transfer dyes have been used to make dye sets thatenhance signals by 2- to 10-fold and simplify the optical configuration.

[0005] Automated fluorescent capillary array electrophoresis (CAE) DNAsequencers appear to be the consensus technology to replace slab gels.Capillary gel electrophoresis speeds up the separation of sequencingproducts and has the potential to dramatically decrease sample volumerequirements. The 96-channel CAE instrument, MegaBACE™, which iscommercially available from Molecular Dynamics (Sunnyvale, Calif.), usesa laser induced fluorescence (LIF) confocal fluorescence scanner todetect up to an average of about 625 bases per capillary (Phred 20window) in 90 minute runs with cycle times of two hours. Confocalspatial filtering results in a higher signal-to-noise ratio becausesuperfluous reflections and fluorescence from surrounding materials areeliminated before signal detection at the photomultiplier tube (PMT).Accordingly, sensitivity at the level of subattomoles per sequencingband is attainable. Confocal imaging is also particularly important incapillary electrophoresis in microchip analysis systems where thebackground fluorescence of a glass or plastic microchip may be muchhigher than that of fused silica capillaries. Capillary arrayelectrophoresis systems will solve many of the initial throughput needsof the genomic community for DNA analysis. However, low volume samplepreparation still presents a significant opportunity to increasethroughput and reduce cost.

[0006] While fluorescent DNA sequencers are improving the throughput ofDNA sequence acquisition, they have also moved the throughput bottleneckfrom sequence acquisition back towards sample preparation. In response,rapid methods for preparing sequencing templates and fortransposon-facilitated DNA sequencing have been developed as havemagnetic bead capture methods that eliminate centrifugation.Thermophilic Archae DNA polymerases have been screened and geneticallyengineered to improve fidelity, ensure stability at high temperatures,extend lengths, and alter affinities for dideoxynucleotides andfluorescent analogs. These improvements have resulted in lower reagentcosts, simpler sample preparation, higher data accuracy, and increasedread lengths.

[0007] The sequencing community has also developed higher-throughputmethods for preparing DNA templates, PCR reactions, and DNA sequencingreactions. Sample preparation has been increasingly multiplexed andautomated using 96- and 384-well microtiter plates, multi-channelpipettors, and laboratory robotic workstations. In general, theseworkstations mimic the manipulations that a technician would perform andhave minimum working volumes of about a microliter, although stand-alonemulti-channel pipettors are being used to manipulate smaller volumes.

[0008] A typical full-scale sample preparation method for DNA shotgunsequencing on capillary systems begins by lysing phage plaques orbacterial colonies to isolate subcloned DNA. Because capillaryelectrophoresis is more sensitive to impurities in sequencing reactionsthan slab gels, the subcloned DNA insert is PCR amplified toexponentially increase its concentration in the sample. Next,exonuclease I (ExoI) and arctic shrimp alkaline phosphatase (SAP) areadded to perform an enzymatic cleanup reaction to remove primer andexcess dNTPs that interfere with cycle sequencing. ExoI is used todegrade the single-stranded primers to dNMPs without digestingdouble-stranded products. SAP converts dNTPs to dNMPs and reduces thedNTP concentration from 200 :M, as used for the PCR reaction, to lessthan 0.1 :M for use with fluorescent sequencing. The reaction isperformed at 37EC and then heated to 65EC to irreversibly denature theExoI and SAP.

[0009] Because the PCR amplification produces excess template DNA forcycle sequencing, the ExoI/SAP treated PCR sample can be dilutedfive-fold before cycle sequencing. This reduces the concentration ofcontaminants into a range that causes less interference with CAEanalysis. Cycle sequencing reagents are added, typically withfluorescently labeled dye primers or terminators and the reaction isthermal cycled to drive linear amplification of labeled fragments.Finally, after cycling, the samples are ethanol precipitated, formamideor another denaturant is added, and the sample is electrokineticallyinjected into the CAE system.

[0010] This workflow has resulted in a dramatic improvement in theperformance of the MegaBACE system and currently appears to be themethod of choice for other CAE systems as well. Using actual samplesfrom single plaques and colonies of human genomic random subclones orExpressed Sequence Tags (ESTs), this workflow with linear polyacrylamideas a separation matrix has improved the success rate of samples over 200base pairs from about 60% to 85-90%, and has improved the averagereadlength from about 350 to greater than 500 bases. Furthermore, thismethod has proven to be quite robust.

[0011] While the above sample preparation methods have greatly increasedthroughput, the cost of reagents remains a major component of the costof sequencing. CAE requires only subattomoles of sample. Reducing thereaction volume will therefore reduce the cost of DNA sequencing.However, substantial reductions in reaction volume can only be achievedif satisfactory methods can be developed for manipulating and reactingsamples and reagents. Ideally, such a method would be automated andconfigured in order that multiple samples could be produced at one time.Moreover, it would be desirable to integrate such a method as a modulecapable of interfacing with additional components, such as CAE and adetector for separation and analysis.

[0012] Several devices have been designed to aid in the automation ofsample preparation. For example, U.S. Pat. No. 5,720,923 describes asystem in which small scale cycling reactions take place in tubes withdiameters as small as 1 mm. The tube are subsequently exposed to thermalcycles produced by thermal blocks to effect a desired reaction. Multiplesamples may be processed in a single tube by drawing in small amounts ofsample, each of which are separated in the tube by a liquid which willnot combine with the sample. Fluid moves through the tubes by means of apump. These features are incorporated into a system which automaticallycleans the tubes, moves sample trays having sample containing wells, andbrings the tubes into contact with the wells in the sample trays.

[0013] U.S. Pat. No. 5,785,926 discloses a system for transporting smallvolumes of sample. In this system, at least one capillary tube is usedto transport small amounts of sample. A precision linear actuatorconnected to a computer controlled motor acts as a pneumatic piston toaliquot and dispense liquid using the tube. The sample amount ismonitored by an optical sensor that detects the presence of liquidwithin the capillary segment. The system includes a fluid stationcontaining liquids to be deposited and a positioning device forpositioning the transport capillary.

[0014] U.S. Pat. No. 5,897,842 discloses a system for the automatedsample preparation using thermal cycling. In this system a reactionmixture is pumped into a capillary tube. One end of the tube is sealedusing pressure from an associated pump while the other end is sealed bypressing the tube against a barrier. The pump also serves to move fluidwithin the tube. Once the ends are sealed, the tube is exposed tothermal cycles. In this system a robotic transfer device moves the tubesbetween the sample preparation station where the pump loads thecomponents of the reaction mixture into the tubes and the thermalcycling station.

[0015] There is an additional need for an automated system that is ableto perform small scale thermal cycling reactions in a highly parallelmanner. The system should allow for rapid preparation of cyclingreactions with minimal reagents. The combination of reducing the amountof reagents required for a reaction and reducing the time required for areaction will greatly reduce the overall cost of preparation of cyclingreactions.

[0016] Capillary array electrophoresis systems and capillaryelectrophoresis microchip analytical systems can detect subattomoles ofreaction products. It is one object of the invention to disclose amethod and system for cycling reactions that operate on a submicroliterscale that takes advantage of the high sensitivity of these analyticalsystems. This reduction of reaction volume will lower the reagentrequirements and cost of each reaction. It is a further object toprovide an automated system that is able to reduce the time required forcycling reaction preparation. It is an additional object of theinvention to provide a system that may be integrated with currentanalytical instruments including capillary array electrophoresis systemsand electrophoresis chips.

[0017] It is a further object of the invention to provide an automatedsystem for preparing reactions and filling a reaction container usingcapillary action. This allows metering a quantity of liquid into acapillary tube length of fixed volume without using external force topump liquids. It is a further object to disclose a reagent meteringdevice which also may act as the reaction container. It is also anobject of the invention to provide a system which allow the nanoscalereaction containers to be cleaned and reused, saving material costs.

[0018] It is a further object of the invention to provide a system withhighly parallel processing, allowing greater throughput. Preferably thesystem would match the density of microwell plates. It is also an objectof the invention to have an automated system in which a number ofdifferent cycling reactions could be performed in parallel using asingle temperature regulation source, allowing more efficient use of thethermal cycling apparatus. It is a further object to perform isothermalreactions in a highly parallel manner in submicroliter volumes. It isalso an object of the invention to provide an automated reactionpreparation system that is able to utilize available automation tools bybeing compatible with standard plate size formats.

SUMMARY OF THE INVENTION

[0019] The above objects have been achieved through a system and methodfor preparing cycling reaction mixtures. The system uses a capillarycassette comprised of a number of capillary tube segments arranged inparallel alignment. The tube segments extend through a substrate and aregenerally positioned with uniform spacing. The capillary cassette may beused both to meter reagents and as a reaction chamber in which thereaction is conducted.

[0020] A reaction mixture containing a nucleic acid sample and reactionreagents for performing a thermal cycling reaction (such as thepolymerase chain reaction, ligase chain reaction, or preparing a chaintermination sequencing reaction) is introduced into the capillaries of acapillary cassette. In one embodiment each capillary contains a uniquenucleic acid sample but the same reaction reagents.

[0021] The reaction mixture may be generated in various manners. In onesample preparation method, sample DNA adheres to the interior of thecapillary tubes of the capillary cassette or onto a substrate. Theliquid in which the DNA was suspended may be eliminated from thecapillary or substrate while the nucleic acid is retained, bound to thecapillary or substrate. The reaction reagents may then be introducedinto the capillary or substrate, combining the sample and reactionreagents to form an assay mixture. In another sample preparation method,the capillaries in a capillary cassette or the wells in a multiwellplate are coated with dehydrated reaction reagents. The nucleic acidsample is introduced into the capillaries of the capillary cassette orthe wells of a multiwell plate and the reaction reagents are dehydratedto form a reaction mixture. If the multiwell plate is used, the reactionmixture is subsequently transferred into the capillaries of a capillarycassette. In another sample preparation method, both the reactionreagents and the nucleic acid sample are metered by the capillaries of acapillary cassette. The capillaries are dipped into the wells of asample plate and a fixed amount of fluid (defined by the interior volumeof the capillary) is drawn into the capillary. The volume of liquidmetered by the capillary tubes is dispensed by positive displacement,centrifugal force, or other displacement method into the wells of amicroplate. A capillary cassette is used to meter both the reactionreagents in a similar manner and dispense the metered liquids onto alocation on a substrate combining the sample and reaction reagents toform a reaction mixture. In any of these reaction mixture preparationmethods reaction reagents, nucleic acid sample and assembled reactionmixture are introduced into the capillary tubes of a capillary cassetteor drawn into the capillary cassette by capillary action. Liquids mayalso be introduced into the capillaries by active filling, such as bypressure or vacuum. For example one end of the capillaries may be sealedwith a liquid impermeable (hydrophobic), gas permeable membrane. Byapplying a vacuum force to one side of the membrane, the capillary willfill with liquid to the level of the membrane where hydrophobic forceswill prevent further filling of the capillary.

[0022] The capillary cassette filled with the reaction mixture is nextsealed by pressing the two ends of the capillary tube segments againstdeformable membranes. The capillary cassette with ends sealed againstdeformable membranes is contained within an interior chamber of atemperature cycling device. The temperature cycling device exposes thecontents of the capillaries to thermal cycles, causing the thermalcycling reaction to occur. In one embodiment the thermal cyclingapparatus is an air thermal cycling device. This device receives thecapillary cassette into an interior chamber where the ends of thecapillaries are sealed. The temperature changes occur using rapidlyflowing air. The temperature of the cycling air may be rapidly loweredby venting air to outside the interior cycling chamber. A thermocouplesensor in the air path of the capillary cassette allows for precisemonitoring of the temperature of the reaction mixture. Given the rapidtransfer of heat through the capillary and precise temperature sensingallowed by the thermal couple, rapid reaction times are possible. Thecomplete thermal cycling times needed for 30 cycles of denaturingheating followed by a period of lower temperature for extension of a600-700 base DNA strand are performed in 30 minutes or less and couldtheoretically be effected in as little as 8 minutes. Following aprogrammed number of thermal cycles, the capillary cassette is removedfrom the temperature cycling chamber.

[0023] The reaction mixture is next dispensed from the capillarycassette and transferred onto a substrate. In one embodiment thesubstrate onto which the completed reaction mixture is dispensed is ananalytical chip. Following transfer from the capillary cassette thereaction mixture may be separated and analyzed. Alternatively, thesample may be dispensed into a microplate or other substrate. Thesubstrate may then be placed, manually or by an automated system, in alocation where it may be analyzed by capillary array electrophoresis. Inaddition to electrophoresis, the instant reaction preparation system mayalso be adapted for use in preparing nucleic acid, protein or otherbiomolecules for microarray analysis, mass spectrometry analysis orother analysis methods. The capillary cassette may also be used forconducting ELISA or other assays requiring binding to a substrate.

[0024] The use of the present system allows a simplified transitionbetween nanoscale and larger scale preparation steps. For example thePCR step may be performed on a nanoscale in the capillary cassette ofthe present invention. The resulting products could be dispensed into amicroplate well for enzymatic clean-up on a larger scale. Followingclean-up, the amplified nucleic acid may be again metered into ananoscale capillary cassette for subsequent reaction mixture preparation(e.g. cycle sequencing). This achieves a simple transition method fromnanoscales to larger scales.

[0025] Depositing the reaction mixtures from the capillary cassette intothe wells of a 96 well plate allows subsequent processing by capillaryarray electrophoresis systems. Post reaction processing is alsopossible. This could include depositing the reaction mixture intoethanol to precipitate the DNA fragments produced in the reaction ordispensing the reaction mixture into formamide to denature doublestranded DNA reaction products.

[0026] Following each use, the capillary cassette may be placed into acapillary cassette washer and washed. Following washing, the capillarycassette may be reused.

[0027] The system can be designed with magazines for holding the sampleplates, the multiwell mixing plates, and the plates containing thefinished reactions. This would allow the system to continuously operateand prepare reaction mixtures. In addition, an integrated system with acentral electronic control would allow for a system which maysimultaneously assemble reaction mixtures, perform thermal cycling, andwash capillary cassettes.

[0028] The system is useful in the preparation of sequencing reactions,but may also be used in highly parallel preparation of cell lysing andplasmid extraction, polymerase chain reactions, ligase chain reactions,rolling circle amplification reactions, screening compound libraries fordrug discovery or compound activity, protein digestion/sequencing,ELISA, radioimmunoassays and other chemical or biochemical reactions orassays.

BRIEF DESCRIPTION OF THE DRAWINGS

[0029]FIG. 1 is a schematic of an integrated system for the preparationof cycling reaction products.

[0030]FIG. 2 is a flow chart illustrating the steps in reactionproduction using the present system.

[0031]FIG. 3A is a perspective view of the capillary cassette of thepresent invention.

[0032]FIG. 3B is a perspective view of the capillary cassette insertedinto a capillary cassette holder.

[0033]FIG. 3C is a flexible capillary cassette.

[0034]FIG. 3D illustrates the capillary cassette of FIG. 3C bent to aframe and mating with the wells of an analytical chip.

[0035]FIG. 3E shows a two layer substrate with microchannels containedwithin.

[0036]FIG. 4A illustrates the dispense head for dispensing liquid fromthe capillary cassette.

[0037]FIG. 4B shows an internal cross section of an air displacementdispense head of FIG. 4A.

[0038]FIG. 4C shows the dispense head of FIG. 4A with the dispense headclosed.

[0039]FIG. 5A illustrates a top view of a centrifuge used to move fluidfrom the capillary cassette of FIG. 3A.

[0040]FIG. 5B illustrates a cross-section of a rotor arm of FIG. 5Aholding a swinging microplate bucket.

[0041]FIG. 6 shows a schematic of an air based thermal cycling devicewith the capillary cassette and holder shown in FIG. 3B inserted intothe temperature cycling device.

[0042]FIG. 7A shows an internal cross section of an air based thermalcycler with integrated capillary cassette sealing membranes.

[0043]FIG. 7B shows a detail of the air based thermocycler of FIG. 7A,with the lid raised to illustrate the chamber into which the capillarycassette is inserted.

[0044]FIG. 7C shows a cross section of the cassette compartment with thecapillary cassette inserted into the internal chamber of the thermalcycler of FIG. 7A.

[0045]FIG. 8A is a front view of the capillary cassette wash station.

[0046]FIG. 8B is a side view of the view of FIG. 8A with the washmanifold lowered and the wash rank raised.

[0047]FIG. 8C is the view of FIG. 8B with the wash manifold raised andthe wash tank lowered.

[0048]FIG. 8D is an interior cross-section of the wash manifold.

[0049]FIG. 8E is a schematic plumbing diagram of the wash station.

[0050]FIG. 8F is a top perspective view of the wash tank.

[0051]FIG. 9 shows a histogram of the percent success versus readlengthwindow for the sequencing analysis of example 2.

[0052]FIG. 10 is an electropherogram of the reaction products of example2.

[0053]FIG. 11 shows a histogram of the percent success versus readlengthwindow for the sequencing analysis of example 3.

[0054]FIG. 12A shows a scanned gel image of electrophoreticallyseparated PCR products prepared at full volume.

[0055]FIG. 12B show a scanned gel image of electrophoretically separatedPCR products prepared at nanoscale (500 nL).

[0056]FIG. 13 is an electropherogram of analysis of prepared sequencingmixtures.

[0057]FIG. 14 is a graph comparing signal strength of reaction productsprepared in tubes, capillaries, and capillaries using surface binding.

DETAILED DESCRIPTION OF THE INVENTION

[0058] In the present invention, it was realized that a capillarysegment could be used both to meter reagents and as a reaction containerfor preforming temperature cycling reactions. The length of thecapillary and the internal diameter (ID) of the bore of the capillarytube define the volume of the interior of the capillary tube segment.Capillaries with a 50-150 um ID are commonly available. The smallinternal diameter of the capillary tubes allows creation of a reactioncontainer having an interior volume less than one microliter. With thepresent invention, capillaries with interior volumes from 10-500nanoliters are adaptable to the preparation of DNA cycle sequencingreactions or any other reaction.

[0059] The process carried out by the present automated system is shownin the flow chart of FIG. 2. The process begins by the assembly of thereaction mixture, box 52, by combination of reagents and a samplenucleic acid. The combined reagents are then introduced into thecapillaries of a capillary cassette, box 54. The ends of the capillariesare next sealed, box 56. The sealed capillary segments are exposed tothermal cycles, box 58, which effect the cycling reaction. Thecapillaries of the capillary cassette are then dispensed onto asubstrate, box 60. The substrate is then transferred to an analyticalsystem for analysis of the reaction mixture, box 62. Details of thisprocess and the structure of the apparatus for carrying out this processare detailed herein.

[0060] In reference to FIG. 1, an automated system is shown for assemblyof reaction mixtures, temperature cycling to effect the chemicalreaction, and dispensing the volume of the completed reaction mixtureonto a substrate for subsequent analysis. In the system an automatedrobot 102 may move the length of stage 114 and may rotate such thatautomated robot 102 may be moved in relation to other components of theautomated system. The automated robot 102 may be rotated to allow thetransfer head 104 on automated robot 102 to access objects on all sidesadjacent to stage 114. The assembly of a reaction mixture would begin bythe transfer head 104 picking up a capillary cassette from cassettehotel 106.

[0061] Capillary cassette 15 is shown in FIG. 3A. The capillary cassetteis comprised of a number of capillary tubes 12 extending through asubstrate 10. It is preferred that the capillary cassette have at leastone row of eight capillary tubes and that the capillary tubes have equalspacing. The capillary cassette shown has substrate 10 with 96 capillarytubes arranged in an 8 by 12 array, with spacing of the tubes matchingthe spacing of the wells of a 96 well microplate. The length ofcapillary tubes 12 extending from either side of substrate 10 isunequal. It is preferred that the shorter end of capillary tube segments12 be shorter than the depth of a microplate well. This allows the shortend of capillary tubes 12 to be inserted into the wells of a microplatewhile substrate 10 rests on the top of the microplate.

[0062] The capillary tubes may be made of any material compatible withthe assay and preparation to be performed, but preferred capillarymaterials include, but are not limited to, glass and silica capillaries,although plastic, metals and other materials may also be used. Capillarytubes of various dimensions may be used, such as 75 um ID capillarytubes or 150 um ID/360 um O.D. capillary tubes.

[0063] The capillary tubes 12 extend through a substrate 10 andpreferably are arranged in a uniform pattern. The capillary tubes are ofequal length and extend through the substrate in a substantiallyparallel orientation such that each of the two opposing ends of thecapillary tubes 12 are coplanar and the planes defined by the ends ofthe capillary tubes 12 are substantially parallel to the substrate 10.The spacing of the capillary tubes may be uniform and selected to matchthe center to center spacing of wells on a microplate. For example on astandard 96 well microplate the capillary tubes would be arranged with a9 mm center to center spacing, on a 384 well microplate the capillarytubes 12 would be arranged with a 4.5 mm center to center spacing.Higher density capillary formats, compatible with 1536 well microplatesor plates with even higher well density, should also be possible. Thecapillary tubes 12 are preferably secured within the substrate such thatthe length of capillary tubes 12 extending from one side of thesubstrate 10 are shorter than the length of the capillary tube on theopposite side of substrate 10. The length of the capillary tubes 12 onthe shorter side of the substrate may be matched to the depth of wellsin a microplate, such that the length of the shorter side is a shorterlength than the depth of a well in a microplate. This feature enablesthe capillary cassette to be inserted into a microplate such that thesubstrate 10 rests against the top lip of the multiwell plate and thecapillaries on one side of the substrate may extend into the multiwellplate without touching the bottom. For example, in a 96 well microplatethe capillary tubes may be disposed on a substrate such that the shorterside of the capillary tube extending from the substrate may be insertedinto wells in a microplate without the capillary touching the bottom ofthe well. This ensures that liquid dispensed into a well is clear of thecapillary to prevent re-entering the capillary.

[0064] The capillary cassette substrate 10 may be made of a fiberglassboard or other rigid or semi-flexible material. The capillary tubes 12may be inserted through evenly spaced holes in the substrate and securedwith adhesive. In one embodiment, the length and width of the substrateare similar to the length and width of a standard 96 well microplate.This simplifies adapting automated systems designed for manipulation ofmicroplates to handle the capillary cassette.

[0065] In some embodiments it may be advantageous to coat the interiorof the capillary with various surface coatings such as ionic andnon-ionic surfactants. Coatings which may be used include bovine serumalbumin (BSA), glycerol, polyvinyl alcohol and Tween 20. The coatingsare introduced into the capillary and dried onto the interior surface ofthe capillary tube. Alternative-ly, covalent modification of theinterior surface with silanization or Griganard reaction may be desired.For example, covalent modification of capillary tubes interior surfaceswhich reduce electroendoosmosis may also be useful in reducing chargesurface effects between a capillary interior surface and components of areaction mixture. U.S. patent application Ser. No. 09/324,892, herebyexpressly incorporated by reference for all purposes herein, disclosesthe use of acryloyldiethanolamine as a covalent capillary coating withadvantageous alkaline stability. In addition to this coating, acrylimideor other known coatings may also be used to covalently modify capillaryinterior surfaces.

[0066] A. Assembly of Reaction Mixture

[0067] Returning to FIG. 1, the automated system allows for thecombination of reaction reagents and sample DNA using the capillarycassette. The capillary cassette would be taken by transfer head 104from the cassette hotel 106 and brought into contact with the samplescontained in a sample plate at location a. The sample plate is dispensedfrom sample plate hotel 108. The sample would be drawn into thecapillary tubes of the capillary cassette by capillary action. Theinternal volume of the capillary tube is defined by the length of thecapillary tube and its internal diameter. The capillary cassette of FIG.3A acts as a fixed volume parallel pipettor, allowing a number ofcapillary tubes to be filled in parallel. Each capillary tube segmentwill meter a discrete amount of liquid which may be subsequentlydispensed.

[0068] Once one end of each capillary is inserted into the samplecontaining well, a liquid will be drawn into the capillary. This smallamount of sample may be combined with other liquids to form a reactionmixture. The sensitivity of analytical instruments such as a capillaryarray electrophoresis system and the exponential amplification ofreaction mixture products enabled by cycling reactions allow fornanoscale reactions and analysis. Very small scale reactions are able toreliably produce reaction mixture products of sufficient quantity foranalysis on a capillary array electrophoresis system or a capillaryelectrophoresis chip. Significantly less reaction reagents are requiredif a nanoscale reaction mixture is enabled.

[0069] The automated system may be used in various ways to preparereaction mixtures. A few of the many such methods for use of the systemin production of reaction mixtures follow.

[0070] Reaction Mixture Preparation Example 1:

[0071] Metering Reagents with Capillary Cassette and Mixing on aSubstrate

[0072] One method to prepare the reaction mixture is to use the pipettorto separately meter the components of a reaction mixture. For examplefor a PCR mixture, the nucleic acid sample and PCR reagents would beseparately metered and dispensed into a single container combining theliquids. In using the automated system of FIG. 1, the automated robot102 moves transfer head 104 containing a capillary cassette to locationa where a sample plate is located. The ends of the capillary tubes ofthe capillary cassette are dipped into the wells. The capillary tubesfill by capillary action, metering a precise amount of sample. The wellsof sample plate contain the nucleic acid sample. The DNA sample shouldbe sufficiently dilute such that 5-20 ng of DNA is contained in the10-10,000 nL volume metered by each capillary tube segment in thecapillary cassette.

[0073]FIG. 4A shows the capillary cassette transferring fluid samplesfrom a multiwell plate 36 into a capillary cassette 15. The capillarytube segments 12 on capillary cassette 15 are extended into the wells ofmultiwell plate 36. The wells of multiwell plate 36 are conical andliquid in the well will flow to the bottom central area of each well.This allows a small amount of liquid within the well to be positionedsuch that a capillary inserted into the center of the well and above thebottom of the well will contact the liquid. The capillary tube segmentsof the capillary cassette then fill by capillary action with the liquidin the wells. It is preferred that the capillary cassette have capillarytube segments which have the same center to center spacing as the wellsof the multiwell plate containing the DNA samples. In one embodiment thecapillary cassette has the same number of capillary tube segments as thenumber of wells in a multiwell plate holding samples.

[0074] After the capillary cassette is dipped into the nucleic acidsample containing wells, the filled capillary cassette may be moved bytransfer head 104 to a dispensing device location 122. At the dispensingdevice location 122, the sample is dispensed onto a substrate. A cleancapillary cassette is then retrieved and dipped into a sample platecontaining the PCR reagents. As seen earlier, the capillary cassettemeters a precise amount of liquid defined by the interior volume of thecapillary tubes held in the capillary cassette. The metered amount ofreaction reagents may be the same volume as the volume of sampledispensed. The reaction reagents are dispensed from each capillary tubesegment onto locations on the mixing substrate containing the nucleicacid sample.

[0075] The present reaction mixture assembly may be used for assembly ofnumerous types of reactions. The same basic method used to assemble thePCR reaction mixture may be adapted to assembly of a cycle sequencingmixture, rolling circle amplification reaction mixture, or otherreaction mixtures.

[0076] When dispensing the contents into a microplate some care must betaken to mix the sample and reaction reagents in a manner which avoidssplattering. A number of different methods have been envisioned todispense liquid from the capillary cassette.

[0077] Capillary Cassette Dispensing Example 1:

[0078] Centrifugal force

[0079] The first method to dispense the contents of the capillarycassette while avoiding splattering uses a centrifuge to dispense thefluid by centrifugal force. The centrifugal force is applied evenly toall of the capillaries in the capillary cassette such that capillariesindependently dispense into microplate wells. The dispensed liquid isdrawn by centrifugal force to the bottom of wells in the multiwellplate.

[0080] In FIG. 5A, the centrifuge 42 is shown having a swingingmicroplate bucket 43 which may contain a multiwell plate with aninserted capillary cassette. The swinging microplate buckets are held onrotor 41.

[0081]FIG. 5B shows a cross-section of swinging microplate bucket 43.The capillary tubes 12 of the capillary cassette are inserted into wells36 a of multiwell plate 36. The cassette is inserted such that theportion of the capillary tubes 12 extending from the substrate 10 areshorter than the depth of the wells 36 a. As shown in FIG. 5B, thecapillary tube 12 extending from substrate 10 do not reach the bottom ofthe wells 36 a of multiwell plate 36. Microplate swinging bucket 43 iscomprised of an arm 45 and a platform 44. An upper end of arm 45 fitsonto latch head 42 on rotor 41. Microplate 36 is positioned on platform44 of microplate swinging bucket 43. When the centrifuge is in motion,platform 144 rotates on latch head 42 such that the multiwell platefaces the side wall of the centrifuge and the centrifugal force on theliquid in the capillary tubes dispenses the liquid into the bottom ofthe wells 36 a of the multiwell plate 36. When conical shaped wells areused, the centrifugal force will draw the liquids within the well to thewell center, causing the sample to locate at a more precise location Theliquid will be displaced from the capillary at fairly low centrifugespeeds.

[0082] In FIG. 1, a low speed centrifuge may optionally be included inthe automated system at the dispensing device location 122. Automatedrobot 102 uses transfer head 104 to pick up a nanotiter plate dispensedonto location b by nanotiter plate hotel 110. The nanotiter plate istransferred by transfer head 104 to the stage of the low speedcentrifuge. A capillary cassette is filled with samples or reactionreagents as described and is transferred onto the nanotiter plate on thestage of the low speed centrifuge. The plate and cassette are then spunin the centrifuge, dispensing the liquid from the capillaries into thewells of the nanotiter plate. Once the liquid has been dispensed and thecentrifuge has stopped rotating, the capillary cassette may by removedby the transfer head and transferred to the cassette washer 118. Thetransfer head 104 can then pick up a clean capillary cassette from thecapillary cassette hotel 106. The clean capillary cassette can be usedto meter a second liquid reaction component which is similarly dispensedusing the centrifuge. In the automated system the centrifuge includes asensor associated with the rotor used in conjunction with a rotorbraking system to stop the rotor in a position which transfer head 104can access such a sensor could be magnetic, optical, mechanical, or useother known means of sensing rotor position.

[0083] Capillary Cassette Dispensing Example 2:

[0084] Air Displacement

[0085] A second method of dispensing the liquid contained in thecapillary tube segments of a capillary cassette is through the use of anair displacement device. With reference to FIG. 1, a nanotiter platedispensed from nanotiter plate hotel is transferred by transfer head 104to the dispensing device location 122. At this location an airdispenser, such as the one pictured in FIG. 4A-C is located.Subsequently a capillary cassette is retrieved by transfer head 104,filled with either sample from a sample multiwell plate or reactionreagents. The capillary cassette is then moved to the dispensing devicelocation 122 and brought into contact with air displacement head. Thesubstrate of the capillary cassette is placed on a receiving platform onthe air displacement head. Alternatively, the air displacement head maybe joinable to automated transfer robot 102.

[0086] With reference to FIG. 4A, the air displacement head 301 is shownwith a capillary cassette 15 held on bottom plate 302. The bottom plate302 is attached to a manifold assembly by hinge 318. Capillary cassettesubstrate 10 is held on foam rubber pad 304 which is secured onto bottomplate 302. An array of holes 325 extend through foam rubber pad 304 andbottom plate 302 which are spaced to allow the capillary tubes 12 toextend through foam rubber pad 304 and bottom plate 302 when thecapillary cassette is positioned on bottom plate 302. The manifoldassembly of the air displacement head is comprised of an upper housing306, chamber unit 310 and a set of clamps 314. Clamps 314 securemembrane 312 to the lower surface of the chamber unit 310. Membrane 312forms a seal to the top surface of the capillary cassette 15 when themanifold assembly is closed over the cassette. Membrane 312 has holes316 corresponding to capillary 12 positions in the cassette when thecapillary 12 positions in the cassette when the capillary cassette 15 isplaced on bottom plate 302. When the top manifold of air displacementhead 301 is closed against bottom plate 302, capillary tubes 12 arepositioned in capillary tube receiving holes 316 on membrane 312. Whenthe air displacement head 301 is closed it may be secured by latch 322which mates with hole 324 to clamp the capillary cassette between thefoam rubber pad 304 and membrane 312 resulting in a seal between the topsurface of cassette 15 and the membrane 312.

[0087]FIG. 4B illustrates a cross sectional view of displacement head301. Upper housing 306 is constructed of metal, acrylic or other rigidmaterial. Gas input coupler 303 is disposed on upper housing 301. When apressurized gas or vacuum line 305 is attached to gas input coupler 303,a vacuum or pressure force may be introduced into upper chamber 307.Held between upper housing 306 and chamber unit 310 is a gas imperviouselastic membrane 308. The area between elastic membrane 308 and upperhousing 306 defines upper chamber 307. Secured onto clamps 314 ismembrane 312. Membrane 312 is pressed against substrate 10 of acapillary cassette inserted into displacement head 301. Substrate 10 issecured within displacement head 301 by bottom plate 302. Rubber pad 304provides a deformable surface which exerts uniform force pressingsubstrate 10 against membrane 312. Membrane 312 has an array of holes316 which allow the capillaries 12 of the capillary cassette to extendthrough membrane 312. When a capillary cassette is inserted into airdisplacement head 301, the substrate seals holes 316 enclosing lowerchamber 313. When pressurized gas is introduced into chamber 307 by gasline 305, elastic membrane 308 will be pressed into lower chamber 313.Membrane 308 is located between upper chamber 307 and lower chambers313. Membrane 308 serves both as seal for the upper end of chambers 313and the chamber displacement actuator when pressure is applied to theupper chamber 307 through coupler 303. The degree of displacement isdependent on the pressure applied. The resulting air displacement willact to dispense liquid from capillary tubes 12 which extend through thecapillary and into the lower chamber 313. By regulating the amount ofpressure applied through line 305, a consistent displacement force willbe applied to each capillary tube. Given the submicroliter volume of thecapillary tube segments, fluctuations in the amount of dispensingpressure should not adversely affect displacement from the tubes.

[0088]FIG. 4C illustrates the closed air displacement head 301. Upperhousing 306 is pulled toward bottom plate 302 by latch 322 in order tocompress membrane 312 against the top of the capillary cassettesubstrate thereby forming a seal. Clamps 314 secure membrane 312 ontochamber unit 310. Air displacement head 301 is mounted on arm 320. Arm320 may extend from automated transfer robot 102 shown in FIG. 1 or bepositioned at dispense location 122. Pressurized gas may be introducedinto upper housing 306 through gas input couple 303.

[0089] This displacement head provides an individual displacementchamber for each of the capillaries dispensed. Although a 16 capillarycassette is depicted, the displacement head may be constructed todispense capillary cassettes having an array of 96 capillaries orgreater capillary densities. The dispensing force applied to eachcapillary is sufficiently small to allow dispensing directly onto asubstrate with the sample dispensed at a discrete location.

[0090] Air displacement or centrifugal displacement may be used todispense liquid from the capillary tube segments in a capillarycassette. It may also be possible to dispense liquid from the capillarytubes using a bank of syringe pumps, applying pressure through a gaspermeable/liquid impermeable (hydrophobic) membrane, usingelectrokinetic dispensing, or other known dispensing means. The airdisplacement head may also be used to dispense finished reactionmixtures onto a substrate for subsequent analysis.

[0091] Reaction Mixture Assembly Example 2:

[0092] Dehydrated Reagents

[0093] A second method to assemble the reaction mixture is to have theregents required for the reaction stored as a dehydrated coating eitheron the interior of a capillary or on a substrate, such as within a wellof a multiwell plate. If the reaction reagents are dehydrated onto theinterior of capillary tube segments in a capillary cassette, introducinga sample into the capillary would cause rehydration, mixing andformation of the reaction mixture. In a similar manner, if the wells ofa microplate are coated with the dehydrated reaction reagents, adding anucleic acid sample into the wells would bring the reaction reagentsinto solution forming an assay mixture. The sample could be metered witha capillary cassette and dispensed from the capillary cassette by one ofthe methods set out above. The sample would bring the dehydratedreaction reagents into solution and mix with the sample containingnucleic acid by diffusion. This provides a method to assemble thereaction mixture in a very simple manner, potentially without the needto dispense the capillary tubes with a centrifuge or air displacementdevice. This could both simplify the reaction processing system andshorten the reaction assembly time.

[0094] For PCR, a dehydrated reagent mixture is commercially available,sold as Ready-to-Go® (Amersham Pharmacia Biotechnology, Piscataway,N.J.). The stabilized, dehydrated reagents may be coated onto theinterior surface of capillary segments or the interior of the wells of amultiwell plate. The Ready-to-Go® product uses a carbohydrate matrix tostabilize the reaction reagents (DNA polymerase, buffer reagents, dNTPs)in a desiccated state. Bringing the reagents in the Ready-to-Go® mixtureinto solution with the liquid nucleic acid sample and primers insolution produces the final reaction mixture required for the reaction.The combination of the stabilized Ready-to-Go® compounds, the templateDNA, primers, and sufficient water produces a final reaction product. Itis contemplated that reagents for chain termination sequencing reactionscould also be stored in a desiccated state.

[0095] The coating could be applied to surfaces by a number of differentmethods including vapor phase coating, filling a capillary (by capillaryaction, pressure filling, etc.) with the Ready-to-Go® mixture andemptying the bulk phase (under vacuum, pressure or other forces), ordipping a substrate (such as a bead) into the reagents and subsequentlydrying the bead.

[0096] Reaction Mixture Assembly Example 3:

[0097] Nucleic Acid Capture

[0098] A third method of assembly of the reaction mixture is to capturethe sample nucleic acid on the surface of a substrate, such as theinterior of a capillary tube segment. The sample nucleic acid may beattached onto the surface by a number of methods. These include covalentattachment, DNA hybridization, hydrophobic interactions, electric field,magnetic field, or other chemical or physical forces. Once the samplehas been attached, the remaining liquid in which the sample wassuspended may evacuated from the capillary or microchip by chemicalreaction or physical force. Air displacement or centrifugal dispensingmethod may be used to empty the capillary, as can a vacuum. The samplenucleic acid would remain on the surface of the substrate. In thissingle step, the sample nucleic acid may be substantially purified. Thereaction reagents may then be combined with the sample nucleic acid,producing the reaction mixture.

[0099] One method to immobilize the nucleic acid sample is to attach thenucleic acid directly to a surface. This may be done by non-covalentmodification (such as surface treatment with NaSCN, DMSO, etc.) orcovalent linkage. There are a number of different covalent attachmentmethods for DNA known in the art. For example, an amino group can beattached to the deoxyribose base of DNA and incorporated during asynthetic reaction, such as during PCR amplification of a DNA plasmidinsert. The glass or silica of a capillary interior could be silanizedand the amino group on the modified DNA would covalently bond to thesilanized interior of the capillary. Alternatively, other chemistriesare available to covalently immobilize DNA onto a surface. Once the DNAis bound to the surface of a capillary or other substrate, the liquid inwhich the DNA was suspended may be eliminated from the capillary and thecapillary may be filled with reaction reagents.

[0100] An alternative method of attaching a nucleic acid to the interiorof the capillaries of a capillary cassette is through affinitychemistry. One common affinity chemistry procedure labels a biomoleculewith biotin and then binds the biotinylated biomolecules to avidin orstreptavidin. The avidin/streptavidin may be used to link thebiotinylated molecules. Nucleic acid labeled with biotin may besubsequently attached to a surface, such as the interior of a capillarytube. This may be accomplished by binding streptavidin to the interiorof the capillary.

[0101] One example of the use of affinity chemistry for the binding ofDNA to the interior of a capillary is disclosed in U.S. Pat. No.5,846,727, hereby expressly incorporated herein for all purposes. Thisreference describes the binding of DNA to the interior surface of thecapillary tubes. The technique requires primers labeled with biotinwhich are combined with dNTPs, a DNA polymerase, and a reaction buffer.This is combined with template DNA, such as plasmids from a DNA librarywith sub-cloned DNA inserts, to form the reaction mixture. In thepresent invention a microplate may contain 96 or more reaction mixtures,each with a unique plasmid with a subcloned DNA sequence. This reactionmixture could be assembled by the method stated in reaction mixtureassembly example 1: namely the reaction reagents and the plasmid samplecould be separately metered and dispensed into a 384 well microtiterplate. In a microplate well the liquids are combined to form a reactionmixture. The reaction mixture is metered into the capillary tubesegments of a capillary cassette. The PCR reaction may be effected bytemporarily sealing the ends of the capillary tube segments and exposingthe capillary cassette to thermal cycles, as described below. Theresults of the PCR reaction are exponentially amplified copies of thesubcloned plasmid DNA insert containing the biotin labeled primer.

[0102] The template DNA containing the biotin labeled primer may then beimmobilized on the walls of the capillary tubes of a capillary cassette.The immobilization capillary cassette would have capillary tubes withavidin or streptavidin coated onto the interior surface of eachcapillary tube. The chemistry for attachment of avidin/streptavidin maybe that disclosed in, for example, L. Amankwa et al., “On-Line PeptideMapping by Capillary Zone Electrophoresis,” Anal. Chem., vol. 65, pp.2693-2697 (1993). The capillary is filled with(3-aniopropyl)trimethoxysilane (3-ATPS), incubated for 30 minutes, andair dried. The dried capillaries in the capillary cassette are nextfilled with sulfosuccinimidyl-6-(biotinamido)hexonate (NHS-LC biotin)which is again incubated followed by air drying. Avidin or streptavidinin phosphate buffer at 7.4 pH is added to each capillary tube. Theavidin binds to the biotin immobilized on the interior of eachcapillary. The double stranded amplified biotinylated PCR productssuspended in a buffer (e.g. Tris-HCl, or EDTA with either NaCl or LiClat 1-3M added for efficaceous binding) are added to the capillary tubeand incubated for 5-10 min. This results in a capillary wall modified asfollows: capillary wall-Si—C₃H₆—NH—CO-biotinavidin orstreptavidin-amplified oligonucleotide with associated biotin primer.

[0103] Once the DNA is immobilized on the interior surface of thecapillary, the contents of the capillary tube may be dispensed in one ofthe methods described and the DNA would remain bound to the surface ofthe capillary. This removes debris and other impurities from the DNApresenting a rapid and effective method of DNA purification. Thecapillary may be rinsed with a buffer for additional purification. Thedefined area of the interior surface of the capillary provides a knownamount of binding sites for the DNA attachment. This provides a simplemethod for normalization of DNA concentration. The capillary cassettemay then be dipped into wells or a reagent reservoir containing thereagents for cycle sequencing. The cycle sequencing reaction can beperformed by temporarily sealing the ends of the capillary tubes bypressing each end of the capillary tubes against a deformable membrane.The capillary cassette may then be exposed to thermal cycles whicheffect the cycle sequencing reaction.

[0104] In this embodiment biotin, rather than avidin or streptavidin, iscovalently attached first to the capillary wall. This aids in theregeneration of the capillary cassette for subsequent binding reactions.After completing the cycle sequencing reaction, it would be difficult toremove the amplified biotinylated DNA without also denaturing the avidinprotein. By having biotin bound to the interior surface of the capillarythe amplified DNA may be easily removed by filling the capillary withphenol or formamide solution at 65-90 degrees C. This denatures theavidin protein without removal of the biotin bound to the interiorsurface of the capillary. This mixture is then dispensed. The capillarycassette may then again be filled with the avidin containing solutionand reused for binding subsequent biotinylated amplified template DNA.

[0105] Prior to filling, the capillary tube segments of the capillarycassette may be coated with a variety of compounds. Coating the interiorsurface of the capillary tube segments with bovine serum albumin (BSA)or polyvinyl alcohol has been shown to improve performance of somereactions, such as preparation of chain termination sequencingreactions.

[0106] B. Thermal Cycling

[0107] Once the reaction mixture is introduced into the capillary tubesegments of the capillary cassette, the ends of the capillaries of thecapillary cassette are sealed and the capillary cassette is exposed totemperature cycles. The ends of the capillary cassette capillaries aresealed by pressing each of the ends of the capillary tubes against adeformable membrane. Returning to FIG. 1, once the capillary cassettehas been filled with the reaction mixture, the ends of the capillariesare sealed and the capillaries are exposed to thermal cycles in thermalcycling device 116.

[0108] In one thermal cycling device, shown in FIGS. 7A-7C, the thermalcycling device has integrated membranes that seal the ends of thecapillaries and exposes the capillary cassette to thermal cycles. Inthis apparatus the means for sealing the ends of the capillaries in thecapillary cassette is incorporated into the thermal cycling device.

[0109] With reference to FIG. 7A, the capillary cassette 15 is held onlip 280 within internal passageway 256 between deformable membranes 264a and 264 b. As seen in FIG. 7B, deformable membrane 264 a is mounted onplatform 261. Lid 262 is secured on platform 261. Platform 261 isattached by pivot 286 to base 265. Pneumatics 284 a, 284 b are attachedat an upper end to platform 261 at pivot 263. Screw 282 acts as a stopfor platform 261 when platform 261 is lowered onto housing 270,enclosing passageway 256. Diffuser 258 promotes temperature uniformly ofair circulating in internal passageway 256. Thermocouple 260 measurestemperature of the circulating air. The function of pivot 277 and bottommembrane platform 200 is described in conjunction with FIG. 7C.

[0110]FIG. 7C shows a cross section of the capillary cassette holdingchamber with capillary cassette 15 inserted into the internal passageway256. The capillary cassette could be inserted into this area byautomated robot 102 of FIG. 1 after the capillary tube segments havebeen filled with the reaction mixture. Capillary cassette 15 ispositioned such that substrate 10 rests on ledge 280. Capillary cassetteis positioned such that the ends of capillary tube segments 12 aredepressed against top deformable membrane 264 a and bottom deformablemembrane 264 b when upper platform 261 is lowered over the capillarycassette and lower platform 271 is raised. Notches 262 a, 262 b sealalong the side lengths of housing 270 when upper platform 261 is loweredto provide a host retaining seal. Screw 282 acts as a stop for upperplatform 261 to prevent the platform from lowering so far that capillarytube segments are bowed or damaged. Base platform 266 is secured to post273 and secured to housing 270. The lower end of pneumatics, 284 b issecured at a lower pivot 271 a to low platform 271. Extending throughlower platform 271 are shoulder screws 268 which extend through housing270 and stationary platform 266 and are secured to lower platform 200.When upper platform 261 is lowered by pneumatic 284 b lower platform 271is also raised toward housing 270. When pneumatic cylinders 284 b, 284 aare retracted, the pneumatic cylinders move to a vertical orientation.Upper platform 261 is lowered and lower platform 271 is raised slightlyin an arc. Lower platform 271 will arc upward on pivot 277 to move to aposition substantially parallel to platform 261 when pneumatic cylinder284 b is fully retracted. When a capillary cassette 15 is inserted intointernal chamber 258 the ability of platform 200 to “float” on springs275 prevents excess pressure from damaging capillary tubes 12 ormembranes 264 a, 264 b. Platforms 261 and 200 exert 400 pounds persquare inch force on capillary tubes 12 providing sufficient sealingpressure. With upper platform 262 lowered, the capillary tube segments12 are sealed at each end by deformable membranes 264 a, 264 b.Deformable membranes 264 a, 264 b may be made of silicon rubber or otherdeformable material.

[0111] Returning to FIG. 7A, a motor 250 turns shaft 251 which rotatessquirrel cage blower 253. Blower 253 produces air movement throughdiffuser 254 to flow into internal passageway 256. The blower generatessufficient circulation flow that the air flowing through internalpassageway 256 circulates at 2000 feet per minute. Diffuser 254 ensuresthat the heat of the air blown by blower 253 is uniform throughoutpassageway 256. Cone 255 on diffuser 254 aids in mixing the flowing air,promotion temperature uniformity through passageway 256. Diffuser 254acts to ensure an even flow of air through passageway 256 in the regionof the capillary cassette and reduces non-uniformity from wall losseffects in internal passageway 256.

[0112] The internal passageway 256 is defined by outer housing 270.Outer housing 270 is preferably of rectangular cross section andcomprised of sheet metal, plastic or other durable material. Theinternal surface of outer housing 270 at all locations except for inlet278 is lined with thermal foam insulation 272. Insulation 272 preventsexcess heating of outer housing 270 and helps retain heat and aidstemperature uniformity of the air circulating through internalpassageway 256. After flowing through first diffuser 254 the air flowsthrough second diffuser 258. Diffusers 254 and 258 promote uniform airflow and temperature uniformity through internal passageway 256. Pastfirst diffuser 254 internal passageway 256 transitions, to match thedimensions of passageway 256 to accommodate. The heated air flows pastthermocouple 260 which is vertically disposed at the center of internalpassageway 256 just beyond second diffuser 258. Thermocouple 260 acts tomonitor the temperature within internal passageway 256. Thermocouple 260may be a temperature monitoring device inserted into a capillary tubesection which extends through outer housing 270 and through foaminsulation 272. Alternatively thermocouple 260 may be selected such thatit accurately reflects the internal temperature of a capillary tube.

[0113] The air circulating through internal passageway 256 passesthermocouple 260 and flows past the capillary tube segments 12 ofcapillary cassette 15. The ends of the capillary tube segments aresealed at their upper end by deformable membrane 264 a mounted on upperplatform 261 which has been lowered to form an air tight seal withhousing 270. The lower end of capillary tube segments 12 are sealed bydeformable membrane 264 b. Deformable membrane 264 b is mounted onplatform 200 which is secured on a bottom surface by shoulder screws268. Shoulder screws 268 extend through housing 270 and retained byplatform 271. Springs 275 located between platform 200 and platform 271provide a biasing force while allowing for platform 200 to float suchthat deformable membrane is biased against the ends of capillaries 12.The function of double acting pneumatics act to seal lid 262 and applyforce to position platform 271 is described in conjunction with FIG. 7C.Lid 262 fits onto housing 270 such that the sheet metal or othermaterial comprising the edge of lid 262 fits on top of housing 270.Membrane 264 a is mounted on upper platform 261 such that membrane 264 aextends into internal passageway 256 at least far enough that membrane264 a is even with insulation 272. As the air travels past capillarytube segments 12, the length of the capillary tube segments 12 belowsubstrate 10 are rapidly heated and cooled to the temperature of the airrapidly moving through internal passageway 256.

[0114] Door 274 controlled by motor 276 is used in conjunction withthermocouple 260 and heating element 252 to control the temperaturewithin internal passageway 256. When door 274 is closed, the aircirculating within internal passageway will not be exchanged withoutside air. As the air continuously passes over heating element 252 theair is rapidly heated until the air comes to the selected temperature.Once thermocouple 260 senses that the temperature is at a selectedtemperature, heating element 252 may be kept at a lower heat output suchthat the internal temperature is maintained. If the temperature needs tobe rapidly dropped, as in during a thermal cycling reaction, door 274may be moved to orientation 274 a by motor 276 with the door 274 movedinto internal passageway 256, allowing all air which has passedcapillary cassette 15 to be exhausted to outside internal passageway256. It is envisioned that a filter or exhaust duct could be mountedabout door 274 to prevent compounds in the circulating air from beingexhausted to the environment. The rapidly circulating air will bequickly exhausted to outside of the thermal cycler while ambient air isdrawn in through air intake 278. Air drawn into internal passageway 256through intake 278 flows through heater 252. The area through which theair moves is restricted by block 259 positioned above heater 252 withininternal chamber 256. Again the temperature of the air within internalpassageway 256 is monitored by thermocouple 260 and when the desiredtemperature drop has occurred, door 274 will be brought toward housing270, reducing air volume drawn through air intake 278.

[0115] By connecting heating element 252, thermocouple 260 and doormotor 276 to an electronic control system, such as a computercontroller, this thermal cycler may perform precise air temperaturevarying sequences. Additional heat is added when needed by heatingelement 252 and heat is exhausted by opening door 274, with thetemperature result of either action monitored by thermocouple 260.Exhausting circulating air through door 274 allows air within internalpassageway to drop in temperature at a rate greater than 10 degrees persecond. The rapid temperature change combined with the rapid transfer ofheat to or from the capillaries allows for efficient temperature cyclingreactions. For example in reactions using a thermostable polymerase, thedenaturing of nucleic acid strands and the annealing of primer totemplate strands each may take place in one to five seconds. Theextension of the primer will require less time to effect since therapidly circulating air and the thin walls of the capillaries rapidlybring the internal volume of the capillaries to the selectedtemperature. The thin walls of the capillaries and the small capillaryvolume enable a rapid temperature change and heat transfer throughoutthe internal capillary volume. This greatly reduces the preparation timerequired for each reaction, allowing more efficient use of the thermalcycler and greater throughput in sample preparation. Presently, a 30cycle PCR amplification may be performed in under 30 minutes. It shouldbe possible to reduce this time to under 8 minutes.

[0116] Once the thermal cycling reaction is complete, upper platform 261may be raised and capillary cassette 15 removed from internal passageway256. During the temperature cycling process, the liquid within eachcapillary tube segment will expand somewhat and some liquid will leakfrom the capillary and be carried away by the rapidly flowing air.However, such loss is only a few percent of the volume of the capillarytube segment and should not present either a contamination problem orcause enough reaction product loss to materially affect subsequentanalysis. In addition, the portion of capillary tube segments 12 locatedbetween substrate 10 and deformable membrane 264 a will receive onlypoor air flow and will be less likely to rapidly reach the denaturationtemperature. However since this length is short, the failure of thisarea to as rapidly reach the denaturation temperature should notadversely affect the ability of the remaining portion of the capillaryfrom producing sufficient reaction product for subsequent analysis.

[0117] An alternative device for sealing the ends of the capillary is acapillary cassette holder which seals the ends of capillary tubesegments of a capillary cassette. With reference to FIG. 3B thecapillary cassette holder is comprised of a pair of parallel deformablemembranes 14 a, 14 b each secured onto a platform 16 a, 16 b. Thedeformable membranes may be silicon rubber seals, Teflon®, plastics orother resilient, deformable material. A pair of parallel posts 9 extendfrom bottom platform 16 a to top support platform 24 where the posts aresecured by internally threaded nut 18. Post 9 passes through platform 24and nut 18 is retained on an annular lip of platform 24. Shoulder screws20 extend through holes in support 24 and are secured to top platform 16b. Springs 22 bias the top platform 16 b against the ends of capillarytube segments 12 while allowing 16 b to float. The substrate 10 ofcapillary cassette 15 may be designed to have holes which conform to thespacing and dimension of posts 18 such that capillary cassette 15 may bemore easily and securely held within holder 23.

[0118] Once the ends of the capillary cassette are sealed in holder 23,the combined capillary cassette and holder may be exposed to thermalcycles. The holder shown seals 16 capillaries. However, a holder may bedesigned to hold capillary cassettes having 96 capillaries or higherdensities of capillaries. In addition to capillary cassettes, chips ofother substrates may be used as the reaction containers. FIG. 3E shows achip substrate 70 comprised of two bonded substrate layers 72, 74. Onelayer 72 has grooves 76 extending the length of the chip. The affixedtop substrate 72 encloses a capillary dimension passage 76 with oppositeopen ends. A liquid reaction mixture may be introduced into the inclosedpassage. The ends of these passages may be sealed by pressing the endsagainst a deformable membrane, as was done with the capillary cassettes.Temperature cycling may require longer times because of greater massmaterial comprising the chip, but cycling times should still be morerapid than conventional cycling.

[0119] For isothermal reactions, such as rolling cycle amplification,temperature cycling is not required to effect the reaction. Once anisothermal reaction mixture is combined and introduced into a capillarycassette, incubation of the cassette at a reaction temperature willallow the reaction to occur. With reference to FIG. 1, the automatedtransfer device may transfer a capillary cassette into incubator 124where the capillary cassette is incubated at a selected temperature. Aset of deformable membranes may be used to seal the ends of thecapillaries during incubation. As was seen in other system components,incubator 124 may be used at the same time as other system components.

[0120] In the case of PCR or chain termination sequencing reactions itis necessary to expose the reaction mixture to temperature cycles. InFIG. 1 the transfer head 104 moves the capillary cassette intothermocycler 116. The thermocycling device may be any device which canexpose the capillary tube segments of the capillary cassette totemperature cycles. Thermal cycling devices which use water, electricfield, heating blocks, or other means may be used. Alternatively, airbased thermal cycling devices are rapid and adaptable to the low volumecycling of the present invention.

[0121] A thermal cycling device which uses air as the temperaturetransfer medium is shown in FIG. 6. The reaction mixture is contained incapillary tube segments which have a high surface to volume ratio andsmall material thickness. This allows very rapid transfer of heatthrough the walls of the capillary and throughout the liquid reactionmixture. An equilibrium temperature is reached rapidly throughout theliquid in the capillary. The use of air as a heat transfer mediumenables the rapid ramping of temperature in the reaction chamber. Rapidcirculation of the air ensures rapid and more uniform heating or coolingof the capillary segments and their contents.

[0122] The capillary cassette 15 sealed within holder 8 is insertedthrough opening 215 in housing 202 of the air based thermal cycler. Theholder 8 is supported by housing surface 215 of the thermal cyclingchamber 210. The capillary tubes 12 mounted to substrate 10 are exposedto the air of thermal cycling chamber 210 such that the air may freelyflow around capillary tube segments 12. Thermocouple 216 monitors thetemperature of the air moving past capillary tubes 12.

[0123] In the air based thermal cycling device, paddle 208 driven bymotor 206 rapidly circulates air with reaction chamber 210. The air israpidly circulated past the capillaries 12 of capillary cassette 15.Halogen bulb 220 acts as a heat source to heat the air within thethermal cycling chamber 210. To effect a thermal cycling reaction, thecirculating air is held at a selected temperature for a selected periodof time. The thermocouple 216 transmits the temperature of the capillarytube segment 12 to microprocessor 218. To effect the needed temperaturechanges the microprocessor instructs actuator 222 to open door 226allowing air to pass through vent 224. As air passes through vent 224additional air is drawn into the reaction chamber through air inlet 203by fan blade 204. Fan blade 204 is driven by motor 206. The venting ofhot air and replacement with cooler ambient air, combined with the rapidcirculation of air by fan 208, a relatively small thermal cyclingchamber 210 and precise measurement of sample temperatures bythermocouple 216 enables rapid temperature ramping. The time requiredfor effecting the thermal cycles is greatly reduced. A typical thermalcycling reaction requires different temperatures for denaturing ofnucleic acid strands, annealing of a primer, and extension of apolymerase. The denaturing and annealing steps occur rapidly in acapillary tube where the small internal volume of liquid will rapidlycome to equilibrium, while the extension of the DNA molecule takes lessthan 10 seconds for a 500 base extension. The time required for eachthermal cycle of the three temperatures (annealing, extension,denaturing) may be reduced to under 15 seconds by using the rapid heattransfer of the air based thermal cycling apparatus. A program of 30cycles, each cycle exposing the capillary to three temperatures forvarying amounts of time theoretically may be effected in under 8minutes.

[0124] The use of the capillary cassette in combination with an airbased thermal cycler allows additional advantages. The capillarycassette holder temporarily seals the capillary, allowing rapid andsimplified sealing of each capillary tube segment. The capillarycassette contains a number of capillary tubes in parallel arrangement,allowing for more efficient use of the thermal cycler and allowinggreater sample throughput. Once the thermal cycle is completed thecapillary cassette 15 contained with in holder 8 is removed throughopening 215. The capillary cassette 15 is released from the holder andis subsequently dispensed.

[0125] The thermal cyclers of FIGS. 6 and 7A-C were illustrated as beingused with capillary cassettes. The same devices are adaptable to othercontainers with opposing ends. For example, a chip-like substrate with aplurality of passageways extending through the chip (as seen in FIG. 3E)has, like a capillary cassette, evenly spaced opposed open ends. Severalchips could be placed into a thermal cycler with the open endstemporarily sealed and exposed to thermal cycles. The rapid temperaturechanges may be a bit slower due to increased material thickness. Othercontainers with opposing open ends may also be used with eithertemperature cycling device.

[0126] C. Dispensing Completed Reaction Mixture

[0127] Following the completion of the thermal cycling reaction, theprepared reaction mixture is dispensed into a substrate for analysis byan analytical system. As noted above, the capillary cassette may bedispensed by air displacement, centrifugal force, vacuum or any otherdisplacement method. The substrate into which the reaction mixture isdisplaced may be the wells of a multiwell plate, locations on a planarsubstrate, or wells which lead into an analytical chip. The reactionmixture, though small, still may produce enough amplified reactionproduct that dilution is necessary.

[0128] Dispensing Completed Reaction Mixture Example 1:

[0129] Direct Dilution

[0130] In reference to FIG. 1, following completion of the temperaturecycling process, the capillary cassette may be removed from air thermalcycler 116 by transfer head 104. The capillary cassette may be moved bytransfer head 104 to be placed in a plate dispensed from finished samplehotel 112. The plate, located at position c, may be a multiwell platesuch as a 384 well microplate. The wells of the plate contain a dilutionliquid, such as formamide, water, TBE or other selected buffer. Thereaction mixture may be dispensed from the capillary tube segments ofthe capillary cassette by positive displacement, centrifugation, orother dispensing means. The reaction may also be dispensed into asolution for further chemical or biochemical reaction.

[0131] Dispensing Completed Reaction Mixture Example 2:

[0132] Ethanol Precipitation

[0133] Ethanol precipitation may be effected in a dispensing meanssimilar to the means of direct dilution. Transfer head 104 of FIG. 1would again take the capillary cassette from air thermal cycler 116 andplace the short ends of the capillaries in a multiwell plate located atposition c. In this case the wells of the plate would contain anethanol, such as 90% ethanol chilled to 4EC. The reaction mixture wouldbe dispensed from the capillary cassette into the ethanol by centrifuge,positive displacement, or other dispensing method. The ethanol couldthen be removed by aspiration or other means. The precipitated DNA couldthen be resuspended in formamide, water or other suitable diluent. Oncethe sample plate is prepared, by either direct dilution or ethanolprecipitation, the plate may be transferred by transfer head 104 toanalytical stage 120. Analytical stage 120 may feed the sample platedirectly into an analytical device, for example a capillary arrayelectrophoresis system, such as MegaBACE™ produced by MolecularDynamics, Sunnyvale Calif. Alternatively, the analytical stage coulddirect the product to other systems for further processing. It is alsopossible to dispense the samples onto a substrate for mass spectrometryanalysis, calorimetric analysis, or other analytical methods.

[0134] Dispensing Completed Reaction Mixture Example 3: DispenseDirectly into Analytical System

[0135] In the previous two examples the samples were dispensed intomultiwell plates. These plates could then be moved manually orrobotically onto a stage for analysis by an analytical system.Alternatively the capillary cassette could be dispensed directly intothe wells of an analytical device, such as an electrophoresis chip. Forexample a capillary cassette having 16 capillaries disposed in thesubstrate in two parallel rows of eight capillaries may dock with 16wells in an analytical microchip. Such a microchip would have an arrayof analytical lanes in fluid communication with a sample port.

[0136] The capillary cassette may be designed such that the spacing ofthe capillaries matches the spacing of the sample reservoir inlets. Forexample, the capillary cassette illustrated in FIG. 3C includescapillaries 12 extending through flexible strip 11. Flexible strip 11may be used alone or in combination with other such strips. Theorientation of the capillaries in an essentially straight line may bealtered by bending strip 11 to form an arc. FIG. 3D illustrates strip 11but allowing capillaries 12 to mate with input ports which is disposedon a substrate in a circular pattern. The liquid in capillaries 12 maythen be electrokinetically injected or otherwise dispensed fromcapillaries 12 into ports of an analytical chip if an appropriateelectrode array is used. Strip 13 may be positioned in the curvedorientation by pressing strip 13 against a curved form, such as a curvedmetal block. This may be done by an automated strip mover incorporatedinto an automated sample preparation system.

[0137] The capillary cassette could be dispensed by air displacement orother dispensing means preferably selected to minimize splattering andbubble formation. Prior to dispensing the prepared reaction mixture intothe wells for analysis, a small amount of a dilutant could be added toeach analytical microchip well. When the capillary cassette isdispensed, the diluent will dilute the samples in the sample wells. Thesub-microliter volume reaction mixtures prepared in the capillarycassette, such as a DNA sequencing reaction product mixture, can readilybe integrated with the analytical microchip for sequencing.

[0138] D. Washing Capillary Cassette

[0139] Following each use of a capillary cassette, the capillarycassette may be washed and reused. After the contents of the capillarycassette have been dispensed or a capillary cassette has otherwise beenused, the capillary cassette is taken to cassette washer 118 where thecassette is washed. Following washing, the cassette is returned to thecassette hotel 106 where the cassette may be reused.

[0140] With reference to FIG. 8A, capillary cassette washer 410 iscomprised of wash manifold 412 and wash tank stage 416. Between washmanifold 412 and wash tank stage 416 is capillary cassette platform 414.Extending from wash tank stage 416 is leg 419. In this wash system, awash liquid is pumped from one or more of containers 452, 454, 456, 458through respective tubes 1, 2, 3, 4 into respective router inputs 453,455, 457, 459. The router directs the selected wash fluid through routeroutflow 451 through line 451 a into the wash tank 440. The fluid isdrawn from wash tank 440 through capillary tube segments of a capillarycassette. The capillary cassette substrate is held between wash manifold412 and wash tank 440 such that if suction is applied to wash manifold412, wash fluid will be drawn through capillary tube segments from washtank 440. The wash solution is drawn by vacuum through wash manifold 412and into waste receptacle 490.

[0141]FIG. 8E provides a schematic of the working of the wash station.Nitrogen tank 460 provides a pressure source to direct fluid flow.Opening manual valve 462 allows gas to flow through regulator 466 andthrough filter 468. Regulator 466 regulates the pressure from thepressure source. Pressure sensor 464 monitors gas pressure from thenitrogen source, and indicates if gas pressure is below a selectedpressure. The pressurized gas flows through filter 468 into line 470.Pressurized gas line 470 branches into the top of sealed wash bottles471, 472, 473, and 474. The pressurized nitrogen pumps the wash liquidwithin each wash bottle into respective fluid lines 471 a, 472 a, 473 aand 474 a respectively through an intake filter 476 on each of saidrespective fluid lines. Each of the sealed wash solution bottles maycontain a different wash solution, such as water, alcohol, a buffer orother wash solution. Although four wash bottles are illustrated, thesystem is adaptable for use with more or fewer wash fluids. In addition,exchange of wash bottles simply requires venting N2 pressure on bottles471, 472, 473, 474 at value 462, the removal of the cap from theselected bottle and replacement of the cap with attached pressure andfluid lines into a new or refilled wash fluid bottle. Each of the fluidlines 471 a, 472 a, 473 a and 474 a terminate in selector valve 478.According to a preset program, the selector valve routes one of theselected fluids from the input line into valve output line 480. Thevalve output line then transports the pressurized liquid into wash tank440.

[0142] The capillary tubes in the capillary cassette function as aconduit for transport of fluid from the wash tank 440 into the washmanifold interior 425. Vacuum source 496 provides a vacuum force oncevalve 492 is open. When vacuum valve 498 is open, a vacuum force isdirected into waste bottle 490 creating negative pressure within line490 a. When valve 495 is open, suction will be applied through suctionline 490 a, suction line 495 a and suction lines 424 a. As suction isapplied through suction ports 424 by suction lines 424 a the negativepressure through interior wash manifold 425 will draw liquid up throughthe capillary tube segments extending into wash manifold interior 425.The liquid will travel through suction passageways 424, into suctionlines 424 a, past valve 495, through suction lines 495 a and 490 a andinto waste bottle 490.

[0143]FIG. 8D illustrates a view of the wash manifold. The bottom of thewash manifold contains holes 426 into which the capillaries areinserted. Wash manifold interior 425 is comprised of lanes joined at afirst end to suction passageways 424 and at a second end to purgepassageways 423. When suction is applied through line 424 a fluid willbe drawn through capillaries into the lanes comprising interior 425,through passageways 424 and into line 424 a. When the purge valve isopened, air will pass through line 423 a, through passageway 423, intointerior 425, and into passageway 424, clearing interior 425 of anyliquid remaining in interior 425.

[0144] Following a wash procedure, wash tank 440 is lowered relative tothe capillary cassette platform such that the ends of the capillary tubesegments are not in contact with the liquid in wash tank 440. The liquidwithin wash tank 440 is drained through drain 484 which transmits thefluid into drain line 484 a when value 485 is opened and suction isapplied through suction line 490 a. The fluid within wash tank 440 willthen drain into waste bottle 490.

[0145] Before each wash solution is introduced into wash tank 440, washfluid supply line 480 and the wash tank distribution manifold 480 a arepurged to empty the line of any previous liquid. This is effected byopening one of the valves in selector valve 478 and flowing wash fluidthrough supply line 480 and through bleed lines 482. Opening valve 487allows a vacuum force to be transmitted through line 490 a through line488 providing suction which in conjunction with fluid pressure is usedto purge the distribution manifold through bleed lines 482. Once washfluid supply line 480 and distribution manifold are purged, valve 487 isclosed and the wash tank is raised and filled. The fill level of washtank 440 is controlled by a selected wash fluid fill time and wash fluidpressure. Overflow port 486 acts as a safety drain to drain off fluidoverfill. If the fluid level within wash tank 440 is too high, liquidwill flow from wash tank 440 into overflow port 486 and into line 486 a.When valve 487 is open, the suction force from line 490 a and 488 willdraw overflow liquid from overflow port 486 into waste bottle 490.Restriction flow valve 441 limits liquid fluid flow through lines 482.

[0146]FIG. 8F shows the top perspective of wash fluid tank 440. An inputline introduces a wash solution into wash fluid distribution manifold480 a. This manifold supplies wash fluid ports 481 which fill tank 440.The spacing of wash fluid ports 481 aids in uniform filling across thewidth of tank 440. The fill time and fluid pressure regulate the amountof fluid filling tank 440. If excess fluid enters tank 440 it will drainfrom overflow port 486.

[0147] To empty the tank, the tank is lowered by the pneumatics asdescribed, and drain 484 is opened. The shape of tank 440 directs fluidto drain 484 when the end of tank 440 containing drain 484 is lowered.This configuration is designed for efficient filling, emptying andpurging of tank 440 and associated fill lines.

[0148] Again with reference to FIG. 8E, once a wash cycle has beencompleted, any liquid remaining within wash manifold interior 425 may beeliminated by opening valve 491 while suction is applied through themanifold. Opening valve 491 causes a pulse of air to be drawn in throughvent 493. The air is introduced into wash manifold interior 425 throughpurge lines 423 a and is removed by suction lines 424 a. If the manifoldis in contact with a capillaries, the relatively narrow bores of thecapillary cassette provide a limited capacity for drawing air throughthe wash manifold. By opening valve 491, a much greater amount of airmay be drawn through the manifold through purge lines 423 a which have amuch greater capacity for drawing air. This will result in a sudden rushof air drawn through the manifold. This acts to clear the wash manifoldof any liquid remaining within the wash manifold interior 425.Preferably manifold interior 425 is purged before and after raising thewash manifold.

[0149] With reference to FIG. 8B, the wash station 410 is shown in sideview. The capillary cassette platform 414 is mounted on support legs445. The reservoir section, shown in internal cross section has at aback lower end of the reservoir, drain outlet 484. Upwardly positionedfrom the drain outlet at the back wall of the tank is overflow outlet486. Disposed at the front of the reservoir is reservoir bleed outlet446. Each outlet is associated with a respective tube and valve, asdescribed in conjunction with FIG. 8E. Each tube carries liquid flowingfrom an associated outlet when the associated valve is opened and vacuumsource applied.

[0150] Capillary cassette platform 414 is held in a fixed position bysupport legs 445. Extending downward from the front of capillarycassette platform 414 is hinge 418 with pivot 432. Attached to a lowerend of hinge 418 is wash tank stage 416. Extending from below wash tankstage 416 is leg 419 which is attached at a lower end by pivot 443 topneumatic cylinder 429. At the back end of the stationary capillarycassette platform 414, the wash manifold is attached at pivot 420. Whenpneumatic cylinder 429 is extended from the lower end, wash tank stage416 will be lowered in an arc away from stationary capillary platform.This occurs when no pressure is applied to 429 and gravity causes thewash tank stage to pivot down. When pneumatic cylinder 429 is extendedfrom the upper end by applied pressure, wash manifold 412 will be raisedin an arc away from capillary cassette platform 414.

[0151] Disposed above capillary cassette platform 414 is wash manifold412. The wash manifold has a purge passageway 423 disposed at a frontend and a suction passageway 424 disposed toward the back end. Therespective lines carrying air to the manifold or removing gas or liquidsfrom the manifold are described in conjunction with FIG. 8E.

[0152] With reference to FIG. 8C, pneumatic cylinder 429 is shown fullyextended from a lower connection pivot 443 on leg 419, through hole 333in capillary cassette platform 414, to an upper connection at pivot 428on wash manifold 412. The extended height of the wash manifold islimited by plate 430 which is secured to the top of manifold 412. Plate430 abuts pin 422 on capillary cassette platform 414 when the washmanifold is raised to a selected level and prevents the wash manifold412 from being raised beyond this level. When suction is applied to washmanifold interior 425 by applying suction through suction passageway424, fluid is drawn through capillaries 12 from tank 440.

[0153] The front end of capillary cassette platform 414 is joined atpivot 432 to hinge 418 and wash tank stage 416 and the back end ofcapillary cassette platform 414 is joined at pivot 420 to wash manifold412. Extending through capillary cassette platform 414 is cutout 434.The dimensions of cutout 434 are such that capillary cassette 15, whenplaced on capillary cassette platform 414 has associated capillary tubesegments 12 extending through capillary cassette platform 414 while thefour edges of capillary cassette substrate 10 are retained on thecapillary cassette platform 414 on the edge of cutout 434. Alignmentpins may be added to capillary cassette platform 414 to properlyposition the capillary cassette.

[0154] To effect the cassette wash sequence, an electronic controllerimplements a sequence of steps. The electronic controller instructsassociated controlled devices of the wash station to carry out aprogrammed wash sequence. The programmed sequence begins with thecapillary cassette being placed on the capillary cassette stage by therobotic transfer device. The wash manifold lowers onto the capillarycassette such that the shorter end of capillary tube segments extendinto the wash manifold and the opposite end of the capillary tubesegments are within the wash liquid in the wash tank once filled. Thesubstrate provides a partial seal between the wash manifold and cassettesuch that when suction is applied to the capillary tube segments by thewash manifold, fluid will be drawn up into the wash manifold through thecapillary tube segments. The wash solution supply line is purged withthe first selected solution to clear the previous solution from theline. As noted in relation to FIG. 8E, the purge solution is removedthrough distribution manifold to drain 484 and bleed lines 482 to washwaste line 488 and 490 a then into waste bottle 490. The wash tank 440is then raised and filled with the selected wash solution.

[0155] A vacuum is applied to the wash manifold causing the solution inthe wash tank to be drawn up through all of the capillary tube segmentsin the capillary cassette. After the programmed wash duration, the washtank is drained and lowered. The vacuum force is continued through thewash manifold, drawing air through the capillary tube segments. Once thecapillary tube segments are dried, the vacuum line of the wash manifoldis turned off. The wash solution supply line is purged with the nextwash solution and the steps of raising and filling the wash tank,drawing the wash solution through the capillary tube segments andemptying the wash tank are repeated for each selected solution. Thespecified sequence may repeat these steps for any number of washsolutions. After the final wash has been completed and the tank emptied,air is drawn through the capillaries by applying a vacuum to the washmanifold, drying the capillary tube segments. Periodically the purgevalve 491 is opened and air is drawn through vent 493 into purge lines423 a into purge inlets 423. This draws a blast of air through washmanifold interior 425 and clears the wash manifold interior of anyremaining liquid, ensuring that any remaining liquid within the washmanifold will not wick back into the capillaries. The manifold vacuum isthen shut off and the manifold is raised, removing the manifold from thecapillary cassette. The manifold vacuum is again applied and the purgevalve 491 is opened and air is drawn through vent 493 into purge line423 a into purge inlet 423. This ensures that any remaining liquid isremoved from the wash manifold interior. The vacuum is then shut off.The washed and dried capillary cassette may then be moved by thetransfer robot to a capillary cassette hotel or other location.

[0156] System Integration

[0157] The components of the system could be integrated in a combinedsystem which allows several elements of the complete system of FIG. 1 tooperate at the same time. For example, electronic control device 123 maybe used to send instructions to the components of the integrated system.The electronic control device may be a computer which sends electronicsignals to various system components to effect a programmed set ofinstructions. Elements of the system could operate simultaneously,increasing system efficiency. For example automated robot 102 couldretrieve a capillary cassette from cassette hotel 106, place thecapillary cassette in a sample plate at stage a. An amount of samplefrom the plate is drawn into the capillary tubes by capillary action.The capillary cassette could then be moved to be placed on top of ananotiter plate such that the short ends of the capillary tube segmentsare in the wells of the nanotiter plate. The robot 102 could thentransfer the combined nanotiter plate/capillary cassette to dispenselocation 122 for dispensing. The movement of the robot 102, transferhead 104 and dispensing device located at location 122 are controlled byelectronic control device 123.

[0158] At the same time that a reaction mixture is being assembled, theelectronic control device could also be sending electronic signals tothermocycler 116. The vent door, heating element, and thermocouple ofthermocycler 116 could be linked to electronic control device 123,allowing electronic control device 123 to effect a selected temperaturecycling procedure by regulating the temperature at which air is cyclingwithin the thermal cycler. This precise monitoring allows thetemperature cycling procedure to be effected in a minimum amount oftime. Once the thermal cycling procedure is complete, the electroniccontrol device 123 could electronically instruct the thermal cycler toshut off the thermocycler fan and heating element and open the lidpneumatically to allow a capillary cassette to be removed from theinterior of the thermal cycler.

[0159] While automated robot 102 is moving capillary cassettes toassemble a reaction mixture and the thermocycler is operating, thecassette washer 118 could also be cleaning a capillary cassette. Againthe electronic control device 123 could instruct the cassette washer 118to perform a wash sequence in which a capillary cassette is cleaned witha selected sequence of wash liquids and air dried.

[0160] Electronic control device 123 enables each element of the systemto be used with maximum efficiency. A single set of instructions toelectronic control device 123 could allow assembly of the reactionmixture, thermal cycling of the reaction mixture to effect the desiredreaction, dispensing of the completed reaction mixture onto ananalytical substrate, movement of the analytical substrate to a stagefor processing by an analytical instrument, and cleaning of usedcapillary cassettes.

[0161] E. Reaction Preparation Examples

[0162] The following examples illustrate the use of the combinedreaction preparation systems. The examples are representative of themany different types of reactions that could be effected with thedisclosed device or system and are described by 1) dye-primer DNAsequencing, 2) dye-terminator DNA sequencing, 3) PCR amplification, 4)PCR amplification, enzymatic purification, and DNA sequencing, and 5) ageneral enzymatic reaction.

EXAMPLE 1 Dye-primer DNA Sequencing Analyzed by CAE

[0163] Dye-primer sequencing reactions were performed within a capillarycassette comprised of 96 uncoated 2.8 cm long, 150 μm I.D., 360 O.D.fused-silica capillaries. Dye-primer sequencing reactions were performedby amplifying template DNA with emission-specific primers correspondingto ddT, ddA, ddC, and ddG terminated reactions. The amplification oftemplate was performed as single reactions in each capillary and pooledinto a common well for post-reaction processing and analysis. Thecolor-specific primers were based on the M13 -40 FWD primer(5′-FAM-GTTTTCCCAGT*CACGACG-3′), with 5-carboxyfluorescein (FAM) as thedonor dye, and a termination-specific fluor attached to the indicatedthymine (T*) as the acceptor dye. The thymine was labeled with FAM forddC-terminated reactions (C-FAM), 6-carboxyrhodamine for ddA reactions(A-REG), N,N,N′,N′-tetramethyl-5-carboxyrhodamine for ddG reactions(G-TMR), and 5-carboxy-Xrhodamine for ddT reactions (T-ROX). A mastermix for 100 dye-primer sequencing reactions was prepared by combining 65:L reaction buffer (220 mM Tris-HCl, pH 9.5, 33.2 mM MgCl₂), 100 :Ldye-primer solution (either 1 μM T-ROX, 1 μM G-TMR, 0.5 μM A-REG, or 0.5μM C-FAM), 100 ΦL of the corresponding deoxy- and dideoxynucleotide mix(0.94 mM DATP, dCTP, dTTP, 7-deaza-dGTP, with 3.1 uM dideoxynucleotide),10 ΦL of enzyme (32 U/ΦL ThermoSequenase), and 225 ΦL filtered deionizedwater. This solution was aliquoted into a 96-well reagent plate prior tomixing with template DNA. The general mixing scheme required the use oftwo capillary cassettes and a 384-well “mix plate”. The first capillarycassette (transfer cassette) was dipped in a solution of template DNA(20 ng/:L M13mp18), and then inverted onto the top of a 384-well “mixplate” with the short ends of the capillaries inserted into the wells.The inverted transfer cassette and mix plate were placed inside abenchtop centrifuge. A balance plate was added to balance the rotor andthe centrifuge brought to 3,000×g for 5 seconds. The centrifugationuniformly dispensed the contents of the transfer cassette intoindividual wells of the 384-well plate. After the centrifuge step, thetransfer cassette was transferred to the capillary cassette washer 410for cleaning, and the mix plate was used for a subsequent centrifugestep for reagent addition.

[0164] To add reagents, a second capillary cassette, (the reactioncassette), was dipped into the wells containing sequencing reagents(prepared as described in the preceding paragraph) and inverted over thewells of the same 384-well plate. The reaction cassette and mix platewere placed in the centrifuge, spun at 3,000×g for 5 seconds, andremoved from the centrifuge. At this point each well contained 500 nL oftemplate DNA and 500 nL of sequencing reagents to form the finalreaction mixture. The second capillary cassette (used to add reagents)was then dipped into the 1 :L mixture contained in the mix plate,filling the capillaries of the reaction cassette in 500 nL.

[0165] The capillary cassette was inserted into the internal chamber ofan air-based thermal cycler, as described herein, where the ends of thecapillary segments are sealed by depressing the ends of the capillariesagainst deformable membrane. After 30 cycles of 95EC for 2 seconds, 55ECfor 2 seconds, and 72EC for 60 seconds, the thermal cycler was opened,removing the ends of the capillaries from contact with the deformablemembranes. The capillary cassette was removed and placed on top of a384-well “mix plate” with the short ends of the capillaries insertedinto the wells. The capillary cassette and mix plate were placed into acentrifuge, with a balance plate. The reaction products were dispensedby centrifugal force (˜2500 g) into a microtiter plate containing 40 μLof 80% isopropyl alcohol. After an initial reaction, the capillarieswere washed as described herein. After the four dye-primer reactions hadbeen performed in four individual capillary cassettes and the productspooled into the wells of a microtiter plate, the samples weresubsequently centrifuged at 3000×g for 30 minutes. The alcohol wasdecanted by a gentle inverted spin, and the samples were resuspended in5 μL of ddH20 for electrokinetic injection and analysis by capillaryarray electrophoresis.

[0166] Analysis of the DNA sequencing fragments was performed withMegaBACE, a 96-capillary array electrophoresis instrument (MolecularDynamics, Sunnyvale, Calif.) using scanning confocal laser-inducedfluorescence detection. Separations were performed in 62 cm long, 75 μmI.D., 200 μm O.D. fused-silica capillaries with a working separationdistance of 40 cm. Electroosmotic flow was reduced by Grignard couplingof a vinyl group to the capillary surface and acrylamide polymerization.The capillaries were filled with a fresh solution of 3% linearpolyacrylamide (LPA)(MegaBACE Long Read Matrix, Amersham Life Sciences,Piscataway, N.J.) which was pumped through the capillaries underhigh-pressure from the anode chamber to individual wells of a 96-wellbuffer plate contained in the cathode chamber. Each well was filled with100 :L of Tris-TAPS running buffer (30 mM Tris, 100 mM TAPS, 1 mM EDTA,pH 8.0). The matrix was equilibrated for 20 minutes followed bypre-electrophoresis for 5 minutes at 180 V/cm. Prior to sampleinjection, the cathode capillary ends and electrodes were rinsed withddH₂O to remove residual LPA prior to sample injection.

[0167] DNA sequencing samples were electrokinetically injected atconstant voltage from a 96-well microtiter plate according to thespecified conditions; one preferred injection condition for 500 nLsamples is 40 seconds of injection at an applied voltage of 2 kV. Afterinjection, the capillary ends were rinsed with water, the buffer platewas placed in the cathode chamber, and the electrophoresis run wascommenced. Separations were typically for 120 minutes at 8 kV. Computercontrolled automation of the instrument and data collection wasperformed using LabBench software (Molecular Dynamics, Sunnyvale,Calif.). Specific injection and run conditions were tailored to thereaction mixture to be analyzed.

[0168] The reproducibility of the described method for sub-microliterdye-primer cycle sequencing is shown in FIG. 9. This histogram shows thepercent success versus readlength window and shows that the method ishighly reproducible. Over 80 percent of the sequenced DNA inserts had areadlength over 600 bases. Overall, this plate yielded 55,000 highquality bases, with an average readlength of 605 bases.

EXAMPLE 2 Dye-primer DNA Sequencing Analyzed by a CAE Microchip

[0169] In another analysis example, dye-primer reactions performed inthe same capillary cassette were analyzed by direct injection into amicrofabricated “chip-based” analyzer. In this example, a dye-primerreaction terminated by ddT was performed as described and dispensed intothe sample wells of a microchip containing 1.5÷μL of ddH₂O. Theelectropherogram is featured in FIG. 10 exemplifying microchip analysisof reactions performed in the described system.

EXAMPLE 3 Dye-terminator Cycle-sequencing with Alcohol PrecipitationPurification

[0170] Dye-terminator cycle-sequencing was demonstrated using thecapillary cassette system and alcohol precipitation for cleanup prior tocapillary array electrophoresis. In this example, the sequencingreaction mix was prepared by mixing 400 μL of sequencing reagents(Dyenamic ET terminator kit, Amersham Pharmacia Biotech, Part 81600)with 100 mL of 5 pmol/μL of M13 -28 FWD primer (5′-TGT AAA ACG ACG GCCAGT-3′). The reaction mix was distributed in 5 μL aliquots to a 96-well“reagent” plate. Mixing of template DNA and sequencing reagents wasperformed in the same series of steps described in Example 1. A secondcassette was used to transfer 500 nL of sequencing reagents from thereagent plate to the wells of the mix plate. This same cassette was thenfilled by capillary action with the template/reagent mixture.

[0171] The capillary cassette was transferred to the air-basedthermal-cycler where the capillaries were sealed between the deformablemembranes within the thermal cycler. Thermal cycling was achieved with30 cycles of 95° C. for 2 s, 55° C. for 2 s, and 60° C. for 60 seconds.After the thermal cycling, the cassette was removed from the cyclingchamber and the contents of the capillaries dispensed by centrifugalforce (3000×g) into a 96-well plate containing 40 μL of 80% ethanol. Thesamples were centrifuged at 3000×g for 30 minutes. The alcohol wasdecanted by a gentle inverted spin, and the samples were resuspended in5 μL of ddH20 for electrokinetic injection and analysis by capillaryarray electrophoresis. The cleanup of dye-terminator reactions byalcohol precipitation, the reproducibility of the technique, and theapplication to “real-world” templates is represented as a histogram ofpercent success versus readlength in FIG. 11. FIG. 11 demonstratesexcellent readlengths and success rates with M13 subclone insertsprepared from the subclone library of a Mouse bacterial artificialchromosome (BAC).

EXAMPLE 4 Dye-terminator Cycle Sequencing with Size-exclusionPurification

[0172] In another example, dye-terminator reactions were performed in500 nL capillaries as described in Example 3, and the reaction productsdispensed into 15 μL of ddH2O by centrifugal force. The 15 mL sampleswere transferred to a filter plate containing 45 mL of hydrated SephadexG-50. The samples were centrifuged through the Sephadex matrix at 910×gfor 5 minutes and the eluent collected in a clean 96-well injectionplate. The samples were electrokinetically injected without furtherdehydration or processing. For 16 samples, an average readlength of 650bases was obtained demonstrating the compatibility of sub-microliterdye-terminator sequencing with alcohol and size-exclusion purification.

EXAMPLE 5 PCR Amplification of Plasmid Insert DNA

[0173] The present technology uses the disclosed system for thepolymerase chain reaction (PCR) amplification of insert DNA (e.g.subclone inserts from a DNA library). The PCR reaction mixture wasprepared by mixing 5 :L of 10 :M of M13 -40 FWD primer (5′ GTT TTC CCAGTC ACG AC 3′) and 5 :L of 10 NM M13 -40 REV primer (5′ GGA TAA CAA TTTCAC ACA GG 3′) with 25 :L of 10× GeneAmp buffer, 15 :L of 25 mM MgCl₂, 5:L of AmpliTaq Gold, 2.5 :L of 1 mg/mL bovine serum albumin (BSA), and67.5 :L of ddH₂O. This mix was aliquoted in equal volumes to sixteen0.20 mL tubes.

[0174] The reaction was initiated by mixing template DNA with the PCRcocktail using the two-capillary cassette and mix-plate methoddescribed. The transfer cassette was dipped into the glycerol stocksolutions of a subclone library and dispensed by centrifugal force intothe wells of a 384-well plate. A second “reaction” cassette was used totransfer 500 nL of PCR cocktail to the same wells by centrifugal force.The capillaries were subsequently dipped into the combined mixture oftemplate DNA and PCR reagents, filling the capillaries by capillaryaction. Amplification was effected by placing the capillaries into thecycling chamber and thermally cycling with an activation step of 95° C.for 12 minutes followed by 30 cycles of 64° C. for 4.5 minutes and 95°C. for 5 seconds.

[0175] The PCR products were analyzed by agarose gel electrophoresis andcompared with the same subclones amplified by large-volume (25 :L)reactions performed in 0.20 mL tubes. Nanoscale capillary cassettesamples were dispensed into 4.5 :L of ddH2O by centrifugal force.Equivalent volume aliquots of full volume reactions were transferredmanually using a low volume pipettor. To each 5 :L sample, 1 :L of 6×loading dye was added and the sample quantitatively transferred to thewells of an agarose gel. Agarose gel electrophoresis was performed usinga 0.7% agarose gel with 1×Tris-acetate-EDTA buffer, pH 8.0. Samples wereseparated for 40 minutes at 15 V/cm, stained with Sybr Green II(Molecular Probes, Eugene, Oreg.), and imaged using a two-dimensionalfluorescence scanner (FluorImager, Molecular Dynamics, Sunnyvale,Calif.). The scanned gel image is shown in FIGS. 12A and 12B. It can beseen that samples prepared at full-volume (FIG. 12A) and 500 nLamplification (FIG. 12B) have the same molecular weight distribution.This example demonstrates nanoscale sample preparation can be analyzedby traditional macro-scale analysis such as agarose gel electrophoresis.

EXAMPLE 6 PCR Amplification and Cycle-sequencing

[0176] A preferred mode of preparing cycle sequencing samples using thepresent invention is to prepare nanoscale PCR samples in the capillarycassette and related instrumentation, perform macroscale ExoI/SAPreactions, and then perform the cycle sequencing in the capillarycassette and related instrumentation. Nanoscale PCR template preparationfor DNA sequencing was demonstrated by performing PCR amplification fromglycerol stock subclones. Glycerol stock subclones were PCR amplified asdescribed in Example 5. After PCR amplification, the contents of thecapillaries were dispensed by centrifugation into the wells of a 96-wellplate containing 4.5 :L of 7.5 mU of shrimp alkaline phosphatase (SAP)and 37.5 mU of exonuclease I (ExoI). The PCR products and ExoI/SAPsolution were allowed to incubate at 37° C. for 5 minutes to digest theunincorporated primers and to dephosphorylate the unincorporatednucleotides. After an initial incubation, the enzymes were deactivatedby heating the solution to 72° C. for 15 minutes.

[0177] The ExoI/SAP treated PCR products were aliquoted to a fresh384-well mix plate with a transfer capillary cassette and centrifugaldispensing. An equal aliquot of dye-terminator sequencing reagents wereadded to the 500 nL of purified PCR products using another capillarycassette and centrifugal dispensing. The capillaries were then filled bydipping the capillary cassette into the 1 :L reaction mixture. Thetemplate was amplified according to Example 3, dispensed into 40 :L of80% ethanol and purified as described. Analysis of the sequencingreactions was performed by MegaBACE using electrokinetic injection.Portions of six base-called sequencing electropherograms from subclonetemplates prepared by nanoscale PCR amplification from glycerol stocksolutions and by nanoscale cycle sequencing are shown in FIG. 13. Byperforming PCR in a capillary cassette and subsequently transferring thereaction mixture to a microplate, the present system allows a simplifiedtransition from nanoscale (less than 1 μL volumes) to greater thannanoscale reaction volumes. The present system also allows a simplifiedtransition from macroscale (more than 1 μL volumes) to nanoscalereaction volumes, as shown by utilizing the Exo I/SAP reactions forcycle sequencing in the capillary cassette.

[0178] E. Reaction Preparation Examples

EXAMPLE 7

[0179] Isothermal enzyme assay performed in sub-microliter capillarycassette. The use of the described system for performing general enzymereactions was demonstrated with a fluoregenic assay of #-galactosidase.The #-galactosidase (#-Gal) catalyzed hydrolysis ofresorufin-#-D-#-galactosidase (#-Gal) catalyzed hydrolysis ofresorufin-#-D-galactosidase (RBG) was performed within the capillariesof a 96-capillary cassette in which #-Gal hydrolyzes RBG to thefluorophore resorufin.

[0180] A stock solution of 350 micromolar RBG was prepared in 5 mL of100 mM Tris-HCL, 20 mM KCl, and 2 mM MgCl2 to 5 mg of RBG, vortexingvigorously, and filtering the solution through a 0.40 micron filter. Aone-half dilution curve of RBG was prepared from the stock solution. Toeach 10 microliters of RBG solution prepared in 0.20 mL tubes, 200 ug of#-Gal was added and after briefly mixing, filled into a capillarycassette by capillary action. The cassette was placed in an air-cyclerand after 2 minutes at 37 degrees C., the capillary cassette was removedand the contents centrifuged out of the capillaries into a 384-well scanplate containing 5 microliters of 1M sodium bicarbonate. The wells ofthe scan plate were subsequently filled with 50 microliters of ddH20 andthe plate was read by a fluorescent plate reader (Typhoon, MolecularDynamics). A control aliquot from the enzyme reactions performed in the0.20 mL tubes was added to the scan plate.

[0181] Solid-phase capture of the #-Gal was also demonstrated with thissystem by simply filling the cassette with a 20 ug/mL solution of #-Gal,allowing the #-Gal to bind to the capillary surface followed by removingthe excess liquid and drying the cassette using the described cassettewash-manifold. After #-Gal binding the capillaries were filled with RBGsolution by capillary action. The reaction was performed for 2 minutesat 37° C. and analyzed by dispensing into 1M sodium bicarbonate,diluting the water and scanning using a fluorescent plate reader. Theresults are summarized in Figure XYZ showing the expected signal versussubstrate concentration for the tube reactions, and data points ofsignal for the pre-mixed enzyme reaction performed in the capillarycassette, and for the capillary-binding #-Gal assay. This example servesto illustrate the compatibility of the described system for performing arange of general enzyme activity and inhibition assays.

What is claimed is:
 1. A device for dispensing small quantities ofliquid, the device comprising: a capillary cassette, comprised of aplurality of capillary tube sections disposed in an array through asubstrate; and a capillary cassette dispensing means, said dispensingmeans receiving said capillary cassette and dispensing an array ofcapillaries of said capillary cassette to an array of locations.
 2. Thedevice of claim 1, wherein said dispensing means is a pressure drivendispenser.
 3. The device of claim 2, wherein said pressure drivendispenser includes an enclosed area, wherein one end of said capillarytube sections in said capillary cassette may be sealed in said enclosedarea, and wherein the enclosed area enclosing one end of capillaries insaid capillary cassette may be pressurized.
 4. The device of claim 2,wherein said pressure driven dispenser includes a centrifuge.
 5. Thedevice of claim 1, wherein said capillary cassette is an array of 96capillaries.
 6. The device of claim 1, further including a wash stationand a means for transferring said capillary cassette between saiddispensing means and said wash station.
 7. The device of claim 6,wherein said means for transferring said capillary cassette is anautomated transfer device.
 8. The device of claim 7, wherein saidautomated transfer device is a robotic arm.
 9. The device of claim 6,wherein said wash station includes a housing for receiving a capillarycassette and a liquid introduction and removal system that pumps liquidthrough said capillary tube sections and evacuates said liquid from saidcapillary tube sections.
 10. The device of claim 1, wherein eachcapillary tube section of said capillary cassette has an interior volumeof less than one microliter.
 11. A device for transfer of an amount ofliquid from a plurality of locations in a highly parallel manner, thedevice comprising: a capillary cassette comprised of a substrate and atwo dimensional array of capillary tube sections extending through saidsubstrate, each of said capillary tube sections having a first and asecond opposing open ends, said first open ends being coplanar and saidsecond open ends being coplanar; a capillary cassette dispenser; and anautomated capillary cassette transfer device, wherein said capillarycassette transfer device may move said capillary cassette between said afilling location and said capillary cassette dispenser.
 12. The systemof claim 11, wherein said substrate is bendable.
 13. The system of claim11, wherein said substrate is curved.
 14. The device of claim 11,wherein said capillary cassette dispenser employs a pressuredifferential to dispense said capillary tube sections in said capillarycassette.
 15. The device of claim 12, wherein said dispenser is acentrifuge dispenser.
 16. The device of claim 12, wherein said dispenserincludes an enclosed area, wherein one end of said capillary tubesections in said capillary cassette may be sealed in said enclosed area,and wherein the enclosed area enclosing one end of capillaries in saidcapillary cassette may be pressurized.
 17. The device of claim 11,wherein said capillary cassette is comprised of a 8 by 12 array ofcapillary tube sections of equal length extending through a substrate.18. The device of claim 11, further including a wash station, whereinsaid automated capillary cassette transfer device is disposed totransfer said capillary cassette between the capillary cassettedispenser and said wash station.
 19. The device of claim 16, whereinsaid wash station has an enclosure that receives said capillary cassetteand a fluid distribution manifold for introducing fluid throughcapillary tube sections of said capillary cassette.
 20. The device ofclaim 11, wherein each capillary tube section in said capillary cassettehas an interior volume of less than one microliter.
 21. A method todispense small quantities of fluid in a highly parallel manner,comprising: filling an array of capillary tube sections in a capillarycassette with fluid at a filling location, moving said capillarycassette to a dispensing location; and simultaneous dispensing saidcapillary tube sections at said dispensing location.
 22. The method ofclaim 19, further including a step of washing capillary tube sections ofsaid capillary cassette.
 23. The method of claim 20, wherein all stepsare repeated a plurality of times.
 24. The method of claim 19, whereinsaid filling occurs by capillary action without mechanical force. 25.The method of claim 19, wherein said dispensing is effected byestablishing a pressure differential.
 26. The method of claim 23,wherein said dispensing is effected by centrifugal force.
 27. The methodof claim 19, wherein moving said capillary cassette is effected bygripping said capillary cassette by an automated transfer device andmoving said capillary cassette to a programmed location.
 28. The methodof claim 19, where the step of filling said array of capillary tubesections includes filling said capillary tube sections with a volume offluid less than one microliter.
 29. The method of claim 19, wherein allsteps are controlled by a central electronic control.
 30. A device fortransfer of an amount of liquid from a plurality of locations in ahighly parallel manner, the device comprising: a capillary cassettecomprised of a substrate and a linear array of capillary tube sectionsextending through said substrate, each of said capillary tube sectionshaving a first and a second opposing open ends, said first open endsbeing coplanar and said second open ends being coplanar; a capillarycassette dispenser; and an automated capillary cassette transfer device,wherein said capillary cassette transfer device may move said capillarycassette between said a filling location and said capillary cassettedispenser.
 31. The system of claim 30, wherein said substrate isbendable.
 32. The system of claim 30, wherein said substrate is curved.33. The device of claim 30, wherein said capillary cassette dispenseremploys a pressure differential to dispense said capillary tube sectionsin said capillary cassette.
 34. The device of claim 33, wherein saiddispenser is a centrifuge dispenser.
 35. The device of claim 33, whereinsaid dispenser includes an enclosed area, wherein one end of saidcapillary tube sections in said capillary cassette may be sealed in saidenclosed area, and wherein the enclosed area enclosing one end ofcapillaries in said capillary cassette may be pressurized.
 36. Thedevice of claim 30, further including a wash station, wherein saidautomated capillary cassette transfer device is disposed to transfersaid capillary cassette between the capillary cassette dispenser andsaid wash station.
 37. The device of claim 36, wherein said wash stationhas an enclosure that receives said capillary cassette and a fluiddistribution manifold for introducing fluid through capillary tubesections of said capillary cassette.
 38. The device of claim 30, whereineach capillary tube section in said capillary cassette has an interiorvolume of less than one microliter.